Cuypers B, Domagalska MA, Meysman P, Muylder G, Vanaerschot M, Imamura H, Dumetz F, Verdonckt TW, Myler PJ, Ramasamy G, Laukens K, Dujardin JC.

Sci Rep. 2017 Jun 16;7(1):3725. doi:10.1038/s41598-017-03987-0

Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids.

[[!pmidi 28623350 desc="1 µg total RNA reverse-transcribed with SuperScript III and 2 µM random primer (GTATAAGAGACAGNNNNNNN). The RNA strand degraded with 2U RNAse H for 20 mi at 37 °C. The DNA strand was purified with Agencourt AMPure XP beads. 0.6 mM of SL primer (TCAGTTTCTGTA) was annealed to 25 µL of DNA from the previous step in 1x NEB buffer at 98 °C for 5 minutes and cooled down to room temperature for min. Second-strand synthesis with 5U Klenow fragment and 0.4 mM dNTPs in 50 µL at 37 °C for 60 minutes."]]