title: GSM5896846: BASALTAM957; Mus musculus; RNA-Seq
library_construction_protocol: Purified CD4+ T cells were resuspended in 100 μL of Lysis/Binding Buffer (Ambion), vortexed and stored at -80°C until further processing. llumina strand specific mRNA Roughly 100ng of high quality total RNA (RIN >8) was used for the transcriptomic interrogation of control and SIN3A deleted CD4 T cells using Illumina's Stranded mRNA Prep ligation according to the manufacturer's instructions. Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules in the total RNA. The purified mRNA was fragmented and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. A second strand cDNA synthesis step removed the RNA template while incorporating dUTP in place of dTTP in order to preserve strand specificity. Next, double-stranded cDNA was A-tailed, then ligated to Illumina anchors bearing T-overhangs. PCR-amplification of the library allowed the barcoding of the samples with 10bp dual indexes and the completion of Illumina sequences for cluster generation. Libraries were quantified with Qubit dsDNA HS Assay Kit and their profile was examined using Agilent's HS D1000 ScreenTape Assay. Sequencing was carried out in an Illumina NextSeq2000 using paired end, dual-index sequencing (Rd1: 59 cycles; i7: 10 cycles; i5: 10 cycles, Rd2 59 cycles) at a depth of 30 million reads per sample.