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ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Hu KH, Eichorst JP, McGinnis CS, Patterson DM, Chow ED, Kersten K, Jameson SC, Gartner ZJ, Rao AA, Krummel MF.

Nat Methods. 2020 Aug;17(8):833-843. doi:10.1038/s41592-020-0880-2

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Bind antibodies with conjugated oligonucleotides to the cell. Uncage one strand in regions of interest and hybdidise to a specific barclde. Repeat for other ROIs. Dissociate and single-cell sequence.

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Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.

Glasgow E, Kusano K, Chin H, Mezey E, Young WS 3rd, Gainer H.

Endocrinology. 1999 Nov;140(11):5391-401. doi:10.1210/endo.140.11.7136

Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes

cDNA synthesis primed with random hexamers.

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Insights into variation in meiosis from 31,228 human sperm genomes.

Bell AD, Mello CJ, Nemesh J, Brumbaugh SA, Wysoker A, McCarroll SA.

Nature. 2020 Jul;583(7815):259-264. doi:10.1038/s41586-020-2347-0

Insights into variation in meiosis from 31,228 human sperm genomes.

“We identified 813,122 crossovers in the 31,228 gamete genomes.” “Gametes with fewer crossovers in half of their genome tended to have fewer crossovers in the other half of their genome.” “Large concentrations of crossovers in distal regions.” “The sex chromosomes and acrocentric chromosomes had the highest rates of aneuploidy.”

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

Science. 2017 Aug 18;357(6352):661-667. doi:10.1126/science.aam8940

Junyue Cao, Jonathan S. Packer, Vijay Ramani, Darren A. Cusanovich, Chau Huynh, Riza Daza, Xiaojie Qiu, Choli Lee, Scott N. Furlan, Frank J. Steemers, Andrew Adey, Robert H. Waterston, Cole Trapnell, Jay Shendure

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

Methanol fixation.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

Genes Dev. 2018 Sep 18. doi:10.1101/gad.317669.118

Horie T, Horie R, Chen K, Cao C, Nakagawa M, Kusakabe TG, Satoh N, Sasakura Y, Levine M.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

Ptf1a and Meis can reprogram the CNS of Ciona intestinalis into a dopaminergic cell type.

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Ghost cytometry.

Ota S, Horisaki R, Kawamura Y, Ugawa M, Sato I, Hashimoto K, Kamesawa R, Setoyama K, Yamaguchi S, Fujiu K, Waki K, Noji H.

Science. 2018 Jun 15;360(6394):1246-1251. doi:10.1126/science.aan0096

Ghost cytometry.

Single-pixel imaging.

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI).

Chen C, Xing D, Tan L, Li H, Zhou G, Huang L, Xie XS.

Science. 2017 Apr 14;356(6334):189-194. doi:10.1126/science.aak9787

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI).

Tn5 tagmentation with custom adapters ending with T7 promoters, followed by linear amplification by transcription, followed by cDNA synthesis and sequencing.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

Nat Commun. 2018 Feb 12;9(1):619. doi:10.1038/s41467-018-02866-0

Hayashi T, Ozaki H, Sasagawa Y, Umeda M, Danno H, Nikaido I.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

RamDA-seq. First-strand cDNAs are synthesised with a RNAse H minus reverse-transcriptase. DNAse I introduces nicks that prime synthesis of new cDNA molecules. T4gp32 promotes the strand-displacement activity of the reverse-transcriptase. Second-strand cDNAs are synthethised with Klenow fragment (3′ → 5′ exo-) primed with NSRs. In single cells, genomic DNA needs to be removed because of the DNAseI digestion during RT and the low-complexity priming of the second-strand synthesis with NSRs. The resulting DNA molecules are tagmented and sequenced with standard methods. Thus, the method is non-stranded. Despite the use of NSRs, the rRNA rate stays between 20 and 30 %.

Isolation, incubation, and parallel functional testing and identification by FISH of rare microbial single-copy cells from multi-species mixtures using the combination of chemistrode and stochastic confinement.

Lab Chip. 2009 Aug 7;9(15):2153-62. doi:10.1039/b904958d

Liu W, Kim HJ, Lucchetta EM, Du W, Ismagilov RF.

Isolation, incubation, and parallel functional testing and identification by FISH of rare microbial single-copy cells from multi-species mixtures using the combination of chemistrode and stochastic confinement.

Stochastic isolation of single cells into 'plugs' separated by carrier fluid (not emulsions) using a 'chemistrode'.

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scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

Nat Commun. 2018 Feb 22;9(1):781. doi:10.1038/s41467-018-03149-4

Clark SJ, Argelaguet R, Kapourani CA, Stubbs TM, Lee HJ, Alda-Catalinas C, Krueger F, Sanguinetti G, Kelsey G, Marioni JC, Stegle O, Reik W.

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

GpC methyltransferase need accessibiltiy to the native chromatin. RNA and DNA are split primor amplification. SMART-seq on RNA and bisulfite (scBS) on DNA. Throughput limited by the DNA/RNA separation.

Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development.

Development. 2017 Aug 29. pii: dev.151142. doi:10.1242/dev.151142

Adam M, Potter AS, Potter SS.

Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development.

“ a method for single-cell dissociation using a cold active protease from Bacillus licheniformis, which is a soil bacterium that can grow in a wide range of temperatures and has been isolated from Himalayan glaciers”

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Kv4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons.

J Neurosci. 2000 Jan 15;20(2):579-88.

Tkatch T, Baranauskas G, Surmeier DJ.

Kv4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons.

Accute dissociation of slices before cell isolation. Variablility between RT lots? Evaluation of mRNA abundance through a serial dillution and callibration approach.

Identification of cell-specific messenger ribonucleic acids in oxytocinergic and vasopressinergic magnocellular neurons in rat supraoptic nucleus by single-cell differential hybridization.

Yamashita M, Glasgow E, Zhang BJ, Kusano K, Gainer H.

Endocrinology. 2002 Nov;143(11):4464-76. doi:10.1210/en.2002-220516

Identification of cell-specific messenger ribonucleic acids in oxytocinergic and vasopressinergic magnocellular neurons in rat supraoptic nucleus by single-cell differential hybridization.

Single cell cDNA library construction. 5' poly A added by terminal transferase.

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Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.

Kamme F, Salunga R, Yu J, Tran DT, Zhu J, Luo L, Bittner A, Guo HQ, Miller N, Wan J, Erlander M.

J Neurosci. 2003 May 1;23(9):3607-15.

Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.

Proof of concept and method, from Johnson & Johnson, Acturus, and OmniViz.

Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels.

Genome Res. 2005 Oct;15(10):1388-92.

Bengtsson M, Ståhlberg A, Rorsman P, Kubista M.

Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels.

Geometric mean can be more relevant than arithmetic mean when the expression follows a lognormal distribution.

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Amplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells.

Biotechniques. 2006 Apr;40(4):469-70, 472, 474

Ozawa T, Kishi H, Muraguchi A.

Amplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells.

50 % success on B cells to amplify the Ig variable region with polyG tailing and nested PCR.

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Cell fixation and preservation for droplet-based single-cell transcriptomics.

Alles J, Karaiskos N, Praktiknjo SD, Grosswendt S, Wahle P, Ruffault PL, Ayoub S, Schreyer L, Boltengagen A, Birchmeier C, Zinzen R, Kocks C, Rajewsky N.

BMC Biol. 2017 May 19;15(1):44. doi:10.1186/s12915-017-0383-5

Cell fixation and preservation for droplet-based single-cell transcriptomics.

“For rehydration, cells were either kept on ice after fixation (Fixed) or moved from –80 °C to 4 °C (Fixed 1 or 3 weeks) and kept in the cold throughout the procedure. Cells were pelleted at 1000 to 3000 × g, resuspended in PBS + 0.01% BSA, centrifuged again, resuspended in PBS + 0.01% BSA, passed through a 40- or 35-μm cell strainer, counted and diluted for Drop-seq in PBS + 0.01% BSA as described above.”

Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos.

Genome Biol. 2015 Jul 23;16:148. doi:10.1186/s13059-015-0706-1

Fan X, Zhang X, Wu X, Guo H, Hu Y, Tang F, Huang Y.

Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos.

'Single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq).' Linkers added by poly-a tailing of first-strand cDNAs. Random primers but very low levels of rRNA reads.

A cost effective 5' selective single cell transcriptome profiling approach with improved UMI design.

Nucleic Acids Res. 2016 Dec 9. pii: gkw1242. doi:10.1093/nar/gkw1242

Arguel MJ, LeBrigand K, Paquet A, Ruiz García S, Zaragosi LE, Barbry P, Waldmann R.

A cost effective 5' selective single cell transcriptome profiling approach with improved UMI design.

Backload of barcodes between RT and PCR. Changing the terminal base of the template-switching oligonucleotide from RNA to LNA did not increase performance.

Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.

Science. 2016 Aug 26;353(6302):925-8. doi:10.1126/science.aad7038

Habib N, Li Y, Heidenreich M, Swiech L, Avraham-Davidi I, Trombetta JJ, Hession C, Zhang F, Regev A.

Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.

Tissue extracts stored one day in RNAlater before nucleus extraction. SMART-seq2 protocol in microliter-scale volumes. EdU labelling.

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Selective single cell isolation for genomics using microraft arrays.

Nucleic Acids Res. 2016 Aug 16. pii: gkw700.

Welch JD, Williams LA, DiSalvo M, Brandt AT, Marayati R, Sims CE, Allbritton NL, Prins JF, Yeh JJ, Jones CD.

Selective single cell isolation for genomics using microraft arrays.

Cells are harvested by pushing out the magnetic coating of their microraft. Extensive comparison with the C1 platform. Time-lapse microscopy to select proliferative and non-proliferative cells. Commercially available platform.

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

Leung K, Klaus A, Lin BK, Laks E, Biele J, Lai D, Bashashati A, Huang YF, Aniba R, Moksa M, Steif A, Mes-Masson AM, Hirst M, Shah SP, Aparicio S, Hansen CL.

Proc Natl Acad Sci U S A. 2016 Jul 13. pii: 201520964

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

Spotted cells and reagents with a Scienion sciFLEXARRAYER S3.

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA

Cell. 2015 May 21;161(5):1202-14. doi:10.1016/j.cell.2015.05.002

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

Drop-seq. RT primers directly synthethised on beads with a pool strategy to generate identifiers. 20-30,000 molecules counted; ~6,000 genes detected. Capture rate estimated between 10 and 15 %.

Application of an RNA amplification method for reliable single-cell transcriptome analysis.

Biotechniques. 2015 Sep 1;59(3):137-48. doi:10.2144/000114331

Suslov O, Silver DJ, Siebzehnrubl FA, Orjalo A, Ptitsyn A, Steindler DA.

Application of an RNA amplification method for reliable single-cell transcriptome analysis.

Purification by doing nothing: “We observed a substantial loss of template after the PCI procedure; however, we did notice that the inhibition was greatly diminished when RT tubes were simply stored at 40°C for 2–3 days without any cDNA purification. Therefore, we utilized this approach to reduce PCR inhibition.”

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

Gigascience. 2015 Nov 5;4:51. doi: 10.1186/s13742-015-0091-4. eCollection 2015.

Liang Wu, Xiaolong Zhang, Zhikun Zhao, Ling Wang, Bo Li, Guibo Li, Michael Dean, Qichao Yu, Yanhui Wang, Xinxin Lin, Weijian Rao, Zhanlong Mei, Yang Li, Runze Jiang, Huan Yang, Fuqiang Li, Guoyun Xie, Liqin Xu, Kui Wu, Jie Zhang, Jianghao Chen, Ting Wang, Karsten Kristiansen, Xiuqing Zhang, Yingrui Li, Huanming Yang, Jian Wang, Yong Hou and Xun Xu

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

Describes the MIRALCS platform.

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Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

Tsang JC, Yu Y, Burke S, Buettner F, Wang C, Kolodziejczyk AA, Teichmann SA, Lu L, Liu P.

Genome Biol. 2015 Sep 21;16(1):178. doi:10.1186/s13059-015-0739-5

Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

PCA arranged neatly data according to the cell cycle.

Combining protein and mRNA quantification to decipher transcriptional regulation.

Xu H, Sepúlveda LA, Figard L, Sokac AM, Golding I.

Nat Methods. 2015 Aug;12(8):739-742. doi:10.1038/nmeth.3446

Combining protein and mRNA quantification to decipher transcriptional regulation.

Analysis of > 21,000 loci from 31 drosophila embryos. Cooperative binding of 6 Bcd molecules to the hb locus. Even at highest Bcd concentration, the gene is inactive roughly half of the time.

Structure of silent transcription intervals and noise characteristics of mammalian genes.

Zoller B, Nicolas D, Molina N, Naef F.

Mol Syst Biol. 2015 Jul 27;11(7):823. doi:10.15252/msb.20156257

Structure of silent transcription intervals and noise characteristics of mammalian genes.

One type of promoter alternating directly between on and off phases, and another type with multiple successive off sub-phases, causing the distribution of durations of inactive phases to become peaked instead of exponential.

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Single-cell polyadenylation site mapping reveals 3' isoform choice variability.

Velten L, Anders S, Pekowska A, Järvelin AI, Huber W, Pelechano V, Steinmetz LM.

Mol Syst Biol. 2015 Jun 3;11(6):812. doi:10.15252/msb.20156198

Single-cell polyadenylation site mapping reveals 3' isoform choice variability.

“We observed a mean of 6,980 UMIs (transcript molecules) per cell, stemming from an average of 2,800 genes observed per cell.” Maximum efficiency on ERCC spikes: ~20 %

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Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

Science. 2015 Mar 6;347(6226):1138-42. doi:10.1126/science.aaa1934

Zeisel A, Muñoz-Manchado AB, Codeluppi S, Lönnerberg P, La Manno G, Juréus A, Marques S, Munguba H, He L, Betsholtz C, Rolny C, Castelo-Branco G, Hjerling-Leffler J, Linnarsson S.

Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

~5,000 genes (~30,000 molecules) detected in neurons, ~2-3000 (~10,000) in other cells.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

Macaulay IC, Haerty W, Kumar P, Li YI, Hu TX, Teng MJ, Goolam M, Saurat N, Coupland P, Shirley LM, Smith M, Van der Aa N, Banerjee R, Ellis PD, Quail MA, Swerdlow HP, Zernicka-Goetz M, Livesey FJ, Ponting CP, Voet T.

Nat Methods. 2015 Jun;12(6):519-22. doi:10.1038/nmeth.3370

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

mRNAs captured on oligo-dT-coated dynabeads after lysis in conventional tubes or plates.

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Epigenetics. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing.

Science. 2015 May 22;348(6237):910-4. doi:10.1126/science.aab1601

Cusanovich DA, Daza R, Adey A, Pliner HA, Christiansen L, Gunderson KL, Steemers FJ, Trapnell C, Shendure J.

Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing.

Combinatorial barcoding strategy taking advantage of nuclei as molecular compartments.

A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

Bigdeli S, Dettloff RO, Frank CW, Davis RW, Crosby LD.

PLoS One. 2015 Feb 17;10(2):e0117738. doi:10.1371/journal.pone.0117738

A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

Droplets produced with an aerosol spray. Screening for DNA content after amplification.

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Modeling bi-modality improves characterization of cell cycle on gene expression in single cells.

PLoS Comput Biol. 2014 Jul 17;10(7):e1003696. doi:10.1371/journal.pcbi.1003696

McDavid A, Dennis L, Danaher P, Finak G, Krouse M, Wang A, Webster P, Beechem J, Gottardo R

Modeling bi-modality improves characterization of cell cycle on gene expression in single cells.

333 genes studied in 930 cells with the Hurdle model. Nanostring technology. “the cell cycle explains only 5%-17% of expression variability”

Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells.

Buettner F, Natarajan KN, Casale FP, Proserpio V, Scialdone A, Theis FJ, Teichmann SA, Marioni JC, Stegle O.

Nat Biotechnol. 2015 Jan 19. doi:10.1038/nbt.3102

Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells.

scLVM (single-cell latent variable model) normalises data for cell cycle or other sources of variation.

Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis.

Mahata B, Zhang X, Kolodziejczyk AA, Proserpio V, Haim-Vilmovsky L, Taylor AE, Hebenstreit D, Dingler FA, Moignard V, Göttgens B, Arlt W, McKenzie AN, Teichmann SA.

Cell Rep. 2014 May 22;7(4):1130-42. doi:10.1016/j.celrep.2014.04.011

Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis.

Poor correlation at the mRNA level between Cyp11a1 and Ly6C1 or Ly6C2, but most Cyp11a1-positive cells are Ly6C-positive at the protein level.

Genome sequencing of normal cells reveals developmental lineages and mutational processes.

Behjati S, Huch M, van Boxtel R, Karthaus W, Wedge DC, Tamuri AU, Martincorena I, Petljak M, Alexandrov LB, Gundem G, Tarpey PS, Roerink S, Blokker J, Maddison M, Mudie L, Robinson B, Nik-Zainal S, Campbell P, Goldman N, van de Wetering M, Cuppen E, Clevers H, Stratton MR.

Nature. 2014 Jun 29. doi:10.1038/nature13448

Genome sequencing of normal cells reveals developmental lineages and mutational processes

Genome amplification by organoid culture. Mutation rate is higher at earlier developmental stages.

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Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

Fu GK, Wilhelmy J, Stern D, Fan HC, Fodor SP.

Anal Chem. 2014 Mar 18;86(6):2867-70. doi:10.1021/ac500459p

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

only 14 to 23 % of the cDNAs were template-switched.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

Nat Biotechnol. 2014 Aug 3. doi:10.1038/nbt.2967

Pollen AA, Nowakowski TJ, Shuga J, Wang X, Leyrat AA, Lui JH, Li N, Szpankowski L, Fowler B, Chen P, Ramalingam N, Sun G, Thu M, Norris M, Lebofsky R, Toppani D, Kemp DW 2nd, Wong M, Clerkson B, Jones BN, Wu S, Knutsson L, Alvarado B, Wang J, Weaver LS, May AP, Jones RC, Unger MA, Kriegstein AR, West JA.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

Protocol PN-100-7168

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Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

Science. 2014 Jan 10;343(6167):193-6. doi:10.1126/science.1245316

Deng Q, Ramsköld D, Reinius B, Sandberg R.

Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

“we analyzed mean gene expression levels in cells with biallelic expression and indeed observed them to be 2.0 ± 0.1 times higher [95% confidence interval (CI), bootstrap] than the levels in cells with monoallelic expression at all developmental stages”

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Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.

Development. 2014 Jul;141(14):2770-9. doi:10.1242/dev.108910

Abranches E, Guedes AM, Moravec M, Maamar H, Svoboda P, Raj A, Henrique D

Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.

Nanog VNP timelapse shows decorelation between sister cells, suggesting stochastic transitions. Fluctuations were not correlated with the cell cycle.

Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing.

Liang H, Esposito A, De S, Ber S, Collin P, Surana U, Venkitaraman AR.

Nat Commun. 2014 Jun 4;5:4048. doi:10.1038/ncomms5048

Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing.

Cells overcome the G2 checkpoint after accumulating PLK1 activity even if double-strand breaks remain.

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.

Wang BL, Ghaderi A, Zhou H, Agresti J, Weitz DA, Fink GR, Stephanopoulos G.

Nat Biotechnol. 2014 May;32(5):473-8. doi: 10.1038/nbt.2857

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.

Screening genomic clones detected a tandem expansion of the XYLA gene.

Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.

Jaitin DA, Kenigsberg E, Keren-Shaul H, Elefant N, Paul F, Zaretsky I, Mildner A, Cohen N, Jung S, Tanay A, Amit I.

Science. 2014 Feb 14;343(6172):776-9. doi: 10.1126/science.1247651.

Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.

Amplification by in vitro transcription. Many cells were shallow-sequenced.

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Lovatt D, Ruble BK, Lee J, Dueck H, Kim TK, Fisher S, Francis C, Spaethling JM, Wolf JA, Grady MS, Ulyanova AV, Yeldell SB, Griepenburg JC, Buckley PT, Kim J, Sul JY, Dmochowski IJ, Eberwine J.

Nat Methods. 2014 Feb;11(2):190-6. doi: 10.1038/nmeth.2804

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

More genes detected in cultured cells than in in vivo cells.

Image-based transcriptomics in thousands of single human cells at single-molecule resolution.

Battich N, Stoeger T, Pelkmans L.

Nat Methods. 2013 Oct 6. doi: 10.1038/nmeth.2657

Image-based transcriptomics in thousands of single human cells at single-molecule resolution.

« for most genes in a nonsynchronized unperturbed HeLa cell line, at least 1,000 single cells must be sampled to obtain reproducible single-cell distributions of transcript copy number »

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From cellular characteristics to disease diagnosis: uncovering phenotypes with supercells.

Candia J, Maunu R, Driscoll M, Biancotto A, Dagur P, McCoy JP Jr, Sen HN, Wei L, Maritan A, Cao K, Nussenblatt RB, Banavar JR, Losert W.

PLoS Comput Biol. 2013 Sep;9(9):e1003215. doi: 10.1371/journal.pcbi.1003215

From cellular characteristics to disease diagnosis: uncovering phenotypes with supercells.

On marker indicates disease, and another marker classifies it.

Real-time dynamics of RNA polymerase II clustering in live human cells.

Cisse II, Izeddin I, Causse SZ, Boudarene L, Senecal A, Muresan L, Dugast-Darzacq C, Hajj B, Dahan M, Darzacq X.

Real-time dynamics of RNA polymerase II clustering in live human cells.

Short half-life support de novo self-organisation. Inhibition of P-TEFb by flavopiridol stabilises the clusters, suggesting they are made of pre-initiation or pre-elongation complexes.

Transcriptional burst frequency and burst size are equally modulated across the human genome.

Dar RD, Razooky BS, Singh A, Trimeloni TV, McCollum JM, Cox CD, Simpson ML, Weinberger LS.

Proc Natl Acad Sci U S A. 2012 Oct 23;109(43):17454-9. doi: 10.1073/pnas.1213530109

Transcriptional burst frequency and burst size are equally modulated across the human genome.

Large scale analysis of 8,000 loci by random insertion of lentiviral vectors.

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Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation.

Eun YJ, Utada AS, Copeland MF, Takeuchi S, Weibel DB.

ACS Chem Biol. 2011 Mar 18;6(3):260-6. doi: 10.1021/cb100336p

Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation.

Bacteria cultured in agarose droplets before FACS-sorting. Gives details on how to extract droplets from oil phase.

Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

Shalek AK, Satija R, Adiconis X, Gertner RS, Gaublomme JT, Raychowdhury R, Schwartz S, Yosef N, Malboeuf C, Lu D, Trombetta JT, Gennert D, Gnirke A, Goren A, Hacohen N, Levin JZ, Park H, Regev A.

Nature. 2013 May 19. doi: 10.1038/nature12172

Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

After calculating the all pairwise correlations of induced genes, identifies a cluster of 137 coregulated genes.

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Two DNA-encoded strategies for increasing expression with opposing effects on promoter dynamics and transcriptional noise.

Dadiani M, van Dijk D, Segal B, Field Y, Ben-Artzi G, Raveh-Sadka T, Levo M, Kaplow I, Weinberger A, Segal E.

Genome Res. 2013 May 1.

Two DNA-encoded strategies for increasing expression with opposing effects on promoter dynamics and transcriptional noise.

Increasing promoter accessibility increases the rate of activation; increasing the strength of transcription factor binding sites reduce the rate of inactivation.

viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.

Amir EA, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, Shenfeld DK, Krishnaswamy S, Nolan GP, Pe'er D.

Nat Biotechnol. 2013 May 19. doi: 10.1038/nbt.2594

viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.

Non-linear projection and spread of the data, where overlap between dots is reduced. Data layout is reproducible across experiments.

Histone Deacetylase 2 Cell Autonomously Suppresses Excitatory and Enhances Inhibitory Synaptic Function in CA1 Pyramidal Neurons.

Hanson JE, Deng L, Hackos DH, Lo SC, Lauffer BE, Steiner P, Zhou Q.

J Neurosci. 2013 Apr 3;33(14):5924-9

Histone Deacetylase 2 Cell Autonomously Suppresses Excitatory and Enhances Inhibitory Synaptic Function in CA1 Pyramidal Neurons.

Uses a gene gun to manipulate single cells. HDAC2 controls GABA receptor subunits.

Single-cell chemical lysis on microfluidic chips with arrays of microwells.

Jen CP, Hsiao JH, Maslov NA.

Sensors (Basel). 2012;12(1):347-58. doi: 10.3390/s120100347

Single-cell chemical lysis on microfluidic chips with arrays of microwells.

Lyses within 12 s with a strong lysis buffer containing 25 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM sodium pyrophoshpate, 1 mM beta-glycerolphosphate and 2 mM phenylmethylsulfonyl fluoride (PMSF).

Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.

Hocine S, Raymond P, Zenklusen D, Chao JA, Singer RH.

Nat Methods. 2013 Feb;10(2):119-21. doi: 10.1038/nmeth.2305.

Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.

The MDN1 gene in S. cerevisae (14.7 kbp) is transcribed at 25 bases per second in average, but the cell-to-cell variability is high, ranging from 14 to 61 b/s.

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Transcriptome transfer produces a predictable cellular phenotype.

Sul JY, Wu CW, Zeng F, Jochems J, Lee MT, Kim TK, Peritz T, Buckley P, Cappelleri DJ, Maronski M, Kim M, Kumar V, Meaney D, Kim J, Eberwine J.

Proc Natl Acad Sci U S A. 2009 May 5;106(18):7624-9.

Transcriptome transfer produces a predictable cellular phenotype.

Transferred polyadenlyated RNA. Some genes not expressed in the source nor destination cells were induced.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase.

Buganim Y, Faddah DA, Cheng AW, Itskovich E, Markoulaki S, Ganz K, Klemm SL, van Oudenaarden A, Jaenisch R.

Cell. 2012 Sep 14;150(6):1209-22. doi: 10.1016/j.cell.2012.08.023.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase.

48 genes studied by qPCR with Fluidigm's BioMark.

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Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating transcription factor recruitment.

Mol Cell. 2012 Feb 24;45(4):470-82. Epub 2012 Jan 19.

Bumgarner SL, Neuert G, Voight BF, Symbor-Nagrabska A, Grisafi P, van Oudenaarden A, Fink GR.

Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating transcription factor recruitment.

Non-coding ICR1 represses FLO11, but is repressed by its cis-antisense PWR1, resulting in variegated expression.

Transcription of Two Long Noncoding RNAs Mediates Mating-Type Control of Gametogenesis in Budding Yeast.

Cell. 2012 Sep 14;150(6):1170-81. doi: 10.1016/j.cell.2012.06.049. Epub 2012 Sep 6.

van Werven FJ, Neuert G, Hendrick N, Lardenois A, Buratowski S, van Oudenaarden A, Primig M, Amon A.

Transcription of Two Long Noncoding RNAs Mediates Mating-Type Control of Gametogenesis in Budding Yeast.

RME1-induced transcription of the non-coding IRT1 transcript is necessary to repress IME1 and sporulation in haploid yeast.

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Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A.

BMC Mol Biol. 2008 Jul 17;9:63.

Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

PCR noise is much smaller than biological noise for transcripts more abundant than 100 mRNA or 20 cDNA copies. Reverse-transcription is possible in 40 mM guanidine thiocyanate. Reported RT efficiencies vary between 99 and 2 %

Regulation of noise in the expression of a single gene.

Ozbudak EM, Thattai M, Kurtser I, Grossman AD, van Oudenaarden A.

Nat Genet. 2002 May;31(1):69-73. Epub 2002 Apr 22.

Regulation of noise in the expression of a single gene.

Transcription and translation rates varied by point mutations in reporters. Suboptimal translation rates naturally exist in some genes and may be selected for their effect of reducing noise caused by transcription bursts.

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Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.

Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R.

Nat Biotechnol. 2012 Jul 22. doi: 10.1038/nbt.2282.

Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.

If it really uses the SMARTer kit, then their methods section is wrong and they fail to disclose the real sequence of their template-switching oligonucleotide.

Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.

Ståhlberg A, Andersson D, Aurelius J, Faiz M, Pekna M, Kubista M, Pekny M.

Nucleic Acids Res. 2011 Mar;39(4):e24.

Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.

The two subpopulation strongly differ in the (geometric) average of their transcript counts, and the whole population is better described by sum of two simple distributions than by a bimodal distribution.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

Anal Chem. 2012 Apr 17;84(8):3599-606.

Zhang H, Jenkins G, Zou Y, Zhu Z, Yang CJ.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

Primers bound to agarose by 5′ acrydite residues. RT and PCR chained in the same reaction mixture using hot start PCR enzyme. Agarose beads sorted by FACS.

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling.

Bontoux N, Dauphinot L, Vitalis T, Studer V, Chen Y, Rossier J, Potier MC.

Lab Chip. 2008 Mar;8(3):443-50

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling.

For simple RT-PCR, cDNAs were pre-amplified 40× on chip. cDNAs must be dilluted at least 500× before amplification, otherwise TS-PCR is inhibited by leftovers of the RT.

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation.

Proc Natl Acad Sci U S A. 2012 Apr 23.

Muramoto T, Cannon D, Gierlinski M, Corrigan A, Barton GJ, Chubb JR.

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation.

Pulses for developmental genes kept same properties upon induction, while pulses in housekeeping gene varied in duration, frequency, and intensity.

Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

Lavut A, Raveh D.

PLoS Genet. 2012 Feb;8(2):e1002527. Epub 2012 Feb 23.

Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

Direct transport of transcribed RNA to granlues can also account to the discreapancy between RNA and protein counts. Cells unable to localise RNA to granules are more sensitive to gene overexpression.