5 protocols
nucleic acid sequencing protocol
Paired end sequencing at 75bp PE
nucleic acid extraction protocol
Qiagen RNeasy plus mini kit
sample collection protocol
According to supplier recommendations
nucleic acid library construction protocol
Total RNA from each sample was used as input for the Illumina TruSeq Stranded messenger RNA LT Sample Prep Kit (Illumina) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented and copied into first strand complementary DNA using random primers and reverse transcriptase. Second strand cDNA synthesis was then done using DNA polymerase I, RNase H and substituting dUTP for dTTP. The cDNA was ligated to adapters and enriched with PCR to create the final cDNA library. The library was pooled and sequenced
treatment protocol
Dynabeads human T-activator CD4/CD28 beads (Invitrogen, UK) at a ratio of 1 bead:3 cells for 48 hours