GSM5504062: St Foxp3_Scurfy1; Mus musculus; RNA-Seq
Purified CD8+ T cells were resuspended in 100 μL of Lysis/Binding Buffer (Ambion), vortexed and stored at -80°C until further processing. Poly-A RNA was purified and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) using poly-dT oligos carrying a 7 bp-index  and subjected to linear amplification by IVT using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Next, aRNA was fragmented into 250-350 bp fragments with RNA Fragmentation Reagents (Ambion) and dephosphorylated with FastAP (Thermo) following manufacturer´s instructions. Partial Illumina adaptor sequences  were ligated with T4 RNA Ligase 1 (New England Biolabs), followed by a second RT reaction. Full Illumina adaptor sequences were added during final library amplification with KAPA HiFi DNA Polymerase (Kapa Biosystems). RNA-seq libraries quality controls consisted in quantification with Qubit 3.0 Fluorometer (Life Technologies) and size profiles examination with Agilent's 4200 TapeStation System. Libraries were sequenced in an Illumina NextSeq 500 at a sequence depth of 10 million reads per sample.