Instrument: Illumina HiSeq 2000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Total RNA was extracted with Trizol and mRNA was purified using polydT beads. m6A-IP was then performed using an anti-m6A antibody from Synaptic Systems. The immunoprecipitated RNA (IP) and input for each sample was used to generate libraries. 10-25ng of mRNA was used for library preparation using the Illumina TruSeq stranded mRNA kit according to manufacturer’s protocol with two modifications. First, the elute-frag-prime stage was done at 80°C for 2 min to allow annealing without causing fragmentation. RNA was reverse transcribed into first strand cDNA using reverse transcriptase and random primers. Then, the second strand cDNA was synthesized using DNA Polymerase I and RNase H. The cDNA fragments then went through an end repair process with the addition of a single ‘A’ base followed by ligation of the adapters. The products were then purified and enriched by PCR amplification for 10 cycles to generate the final RNA-Seq library. The second modification was done to adjust the bead-DNA ratio to preserve smaller fragments during the adapter ligation double beads clean up step. A beads:DNA ratio of 1:3 instead of 1:1 was used. cDNA libraries were quantified and pooled for cBot amplification and subsequent sequencing on an Illumina HiSeq 2000.