title: LSCs (Leikemia stem cells) Wildtpe 1
design: Total RNA in amount of 0.5~1ug per sample was depleted of ribosomal RNA using the Ribo-Zero kit (Epicentre, Madison, WI). The directional (first strand) cDNA libraries were prepared following the TruSeq Stranded Total RNA Sample Preparation kit manufacturers instructions. RNA was then fragmented at 94oC for 6 min, followed by the first strand cDNA generation. dUTP were incorporated in second strand synthesis in order to effectively quench the second strand during PCR amplification. After adenylation of 3'end and adapter ligation, fragments were selected and enriched with 10 cycles of PCR amplification. Clusters were generated and amplified on single-read flow cells using the Illumina TruSeq SR Cluster Kit v3-cBot-HS according to manufacturer's instructions (Illumina, San Diego, CA). The clusters on flow cell were then sequenced on the Illumina HiSeq2500 using the TruSeq SBS kit v3 (75 cycles). Around 40 million passing filter clusters were generated per sample.