Ribo zero gold rrna removal kit h m r

To perform RNA sequencing, corneal epithelial layer was separated and total RNA was extracted from three independent biological samples. rRNA depletion was performed using Ribo-Zero Gold rRNA removal kit H/M/R (Illumina). Agilent RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent) was used to do total RNA sample QC. Libraries were constructed with a series of standard steps, included RNA fragment and reverse transcription to double-strand cDNA, end repair, tailing and adaptor ligation, PCR amplification, denaturation, and cyclization. The final library was single-strand circle DNA (ssCir DNA) and was amplified with phi29 (Thermo Fisher Scientific) to make DNA nanoball (DNB). DNBs were transformed to single-end 50 bases reads and sequenced on BGISEQ-500 platform (BGI-Shenzhen, China).

Cγ1-cre and Prmt5^F/F Cγ1-cre splenic B cells plated on 40LB cells were separated from the feeder cells by elutriation at day 4 after plating. Total RNA was extracted from four independent biological samples per genotype, using RNeasy plus Mini kit (Quiagen), quality controlled using RNA Pico chip on Bioanalyzer (Agilent) and processed individually for RNA-seq. Samples were depleted of rRNA using Ribo-zero Gold rRNA removal kit H/M/R (Illumina Hu Mm Rt). Libraries were constructed using the KAPA stranded RNA-Seq kit (Roche), which included fragmentation, cDNA, dsDNA, ER, ATL, Ligation and PCR enrichment, and purified by KAPA magnetic beads (Roche). The mean fragment size obtained was 308 bp. QC validation was performed on HSdna chip on Bioanalyzer and Q-PCR titration with NEBnext Library quant Kit for Illumina (NEB). Sequencing PE125 on HiSeq2500 with v4 chemistry (Illumina) at the Genome Center (Quebec).