GSM7635233: V19 cells, WT, 12h 2i treatment, biol rep 1; Mus musculus; RNA-Seq
At specified time points of the time course experiment, media was aspirated from the wells of 12-well plates, followed by a gentle rinse with warm PBS (Corning 21-040-CV) immediate lysis in 1 ml or TRIzol reagent (Thermo 15596026) added directly into the well. Following chloroform addition, the aqueous phase was separated using 5Prime PhaseLock Heavy tubes (QuantaBio 2302830), precipitated with isopropanol, and washed twice with 75% ethanol. Air-dried RNA was resuspended in RNase-free water (Corning 46-000-CM), and 5 μg total RNA per sample were treated with DNAseI using Qiagen RNeasy on-column treatment and cleanup protocol (Qiagen 79254 and Qiagen 74104). 1 μg of DNaseI-treated total RNA was subjected to polyA enrichment using magnetic isolation (NEB E7490), and the mRNA-enriched fraction was converted to barcoded library using NEBNext Ultra II kit (NEB E7770) according to manufacturer's instructions, using Ampure XP SPRI beads (Beckman Coulter A63881) for size selection, and 96 index primers for barcoding (NEB E6609). Molarity of each library was estimated from electrophoresis profile (Agilent Tapestation) and equimolar amounts were combined in one pool. Sample was sequenced in two Illumina NextSeq 500 High Output 75 SR flowcells, demultiplexed, and merged to combine two runs into single .fastq files