GSM4594549: Sequin Mix A replicate 2; synthetic construct; RNA-Seq
Refer to Hardwick et al. 2016. cDNA libraries were constructed with the cDNA-PCR (SQK-PCS109) sequencing and PCR Barcoding (SQK-PBK004) kits using the supplied protocol. RNA sequins are provided in two mixes (A and B), which contain the same transcripts, but at variable molar ratios to parallel differences in gene expression and alternative splicing. Briefly, duplicate libraries of each mix (A1, A2, B1, B2), were constructed using 15 ng as input for cDNA synthesis. Samples were barcoded (1 – 4) using the supplied PCR barcodes (ONT). Transcripts were amplified by 14x cycles of PCR with a 6 min extension time. Sequencing libraries were individually purified using 0.8x AMPure XP beads (Beckman Coulter) and quantified using a Qubit 4.0 (ThermoFisher Scientific). Equimolar amounts of each sample were pooled to a total of approximately 160 fmol (assuming median transcript size = 1 kb), and the pooled library QC'd using a TapeStation 4200 (Agilent Technologies). The final library was loaded onto a R9.4.1 MinION flow cell and sequenced for 65 hrs, with a buffer refuel at 24 hrs with 250 uL buffer FB.
GEO Accession
GSM4594549