RNA-Seq analysis of normal vs NMD-inhibited human erythroblasts, Day 16 NMD replicate 1
Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 ( mRNA Isolation, Fragmentation and Priming Total RNA ) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 ( First Strand cDNA Synthesis ), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40ul AMPure XP beads for the 1st bead selection and 20ul AMPure XP beads for the 2nd bead selection (targeting an insert size of 300-450bp and a final library size of 400-550bp);  and in E7420, Section 1.9A ( PCR library Enrichment ), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705-i712).  Individual libraries were normalized to 10nM and eight samples were  pooled per lane. Sequencing was performed at UC Berkeley s QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150bp paired end reads.