title: GSM9386825: CD4 T cells, Wt 1 Treg; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using Rneasy mini kit (qiagen) Libraries were prepared using the Illumina® Stranded TruSeq® RNA sample preparation Kit. Library preparation started with 500ng total RNA. After poly-A selection (poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second-strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (15 PCR cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of the library were pooled. The pools were quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System. The pool was sequenced on an Illumina NovaSeq6000 sequencing instrument with a PE100 protocol.