title: GSM1831943: WT.Nrp1neg.TR#2; Mus musculus; RNA-Seq
library_construction_protocol: For low-input RNAseq, 1,000 Foxp3+Nrp1-CD4+TCRb+ cells were sorted into TRIZOL and RNA was extracted. RNA was reverse-transcribed using ArrayScript (Ambion) using a specific primer containing T7 promoter, the 5’ TruSeq Illumina adapter, a  8-positions with random nucleotide assignment as a unique molecular identifier (UMI), and a oligodT sequence. Second-strand synthesis was performed using the mRNA Second strand synthesis module (NEBNext #E6111L). After cDNA size selection using AMPure XP beads (0.8x and 1x, BeckmanCoulter- A63987), the product was amplified via in vitro transcription (MEGAshortscript, Invitrogen) for 10 hours and then fragmented (Magnesium RNA Fragmentation Module, New England Biolabs). 3’ indexing adaptor was ligated (truncated T4 RNA ligase 2 -Enzymatics), reverse-transcribed (Superscript II, Invitrogen), and amplified by PCR for 15 cycles (HiFi hotstart PCR kit, Kapa). cDNA cleanup and size selection were performed on AMPure XP beads. Libraries were quantitated by BioAnalyzer using the Agilent High Sensitivity DNA Kit (Agilent 5067-4626) and qPCR using Kapa library quantification kits, and sequenced on a MiSeq (nano kit) and HiSeq 2500 (rapid mode).