GSM2425121: Control_2; Mus musculus; RNA-Seq
RNA of CD4 T cells was extracted using the RNeasy Mini Kit from QIAGEN, following the manufacturer’s instructions. RNA samples were quantified by qubit (Life Technologies) and quality by Agilent Bioanalyzer. All samples had RIN above 8. Libraries were prepared using TruSeq Stranded mRNA kit (Illumina). One hundred fifty nanograms from 6 RNA samples were purified for polyA tail containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the RNA was fragmented. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” based was added and adapter ligated followed by purification and enriched with PCR to create cDNA libraries. Final cDNA libraries were size validated using Agilent Bioanalyzer and concentration validated by qPCR. All libraries were normalized to 10nM and pooled together. 10pM of pooled libraries were loaded onto Illumina cBot for cluster generation. Clustered flow cell was then sequenced Pair-end 100 cycles V3 using Illumina HighSeq 2000 to achieve a minimum of ~35 million reads per sample.
GEO Accession
GSM2425121