title: RNA-seq of MHCCLM3 Cas9-SNORD13H cell
design: The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. Then mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA).  During library preparation, RNA is fragmented to ~300 bp using divalent cations at 94C, followed by first-strand cDNA synthesis with SuperScript II Reverse Transcriptase (Invitrogen, CA, USA) and second-strand synthesis with DNA Polymerase I (NEB, MA, USA). An A-base was then added to the blunt ends of each strand, and dual-index adapters were ligated to the fragments. Size selection was performed with AMPureXP beads. The libraries are amplified through 8 cycles of PCR. The average insert size for the final cDNA libraries were 30050 bp. Finally, the libraries were performed the 2150bp paired-end sequencing (PE150) on the illumina Novaseq 6000 platform by LC Bio Technology CO.,Ltd (Hangzhou, China).