GSM6216007
RNA isolation was performed using the Direct-zol™ RNA Miniprep Plus kit (Zymo Research) according to the manufacturers protocols. The yield and the integrity of RNA were determined by spectrophotometer NanoDrop 2000c. After RNA extraction, mRNA was enriched by using Oligo dT beads from the KAPA mRNA Capture Kit (KK8581). Subsequent library preparation steps of fragmentation, adapter ligation and cDNA synthesis were done on the enriched mRNA using the KAPA RNA HyperPrep kit (KK8540). Truncated universal stub adapters were used for ligation, and indexed primers were used during PCR amplification to complete the adapters and to enrich the libraries for adapter-ligated fragments. Samples were checked for quality on an AATI (now Agilent) Fragment Analyzer. Samples were transferred to the Vincent J. Coates Genomics Sequencing Laboratory (GSL), a QB3-Berkeley Core Research Facility at UC Berkeley, where Illumina sequencing library molarity was measured with quantitative PCR with the Kapa Biosystems Illumina Quant qPCR Kits on a BioRad CFX Connect thermal cycler. Libraries were then pooled evenly by molarity and sequenced on an Illumina NovaSeq6000 150PE S4 flowcell, generating 25M read pairs per sample. Raw sequencing data was converted into fastq format, sample specific files using the Illumina bcl2fastq2 software on the sequencing centers local linux server.