GSM5510091: Naïve Control 1; Mus musculus; RNA-Seq
Tregs were isolated by FACS-sorting. Total RNA was extracted by RNeasy Micro Kit (Qiagen) per manufacturer's instructions Libraries were prepared using a SMARTer Stranded Total RNA-Seq v2 – Pico Mammalian kit (Takara) per manufacturer's instructions. Briefly, between 250pg-10ng of total RNA was fragmented and then reverse transcribed into cDNA using random primers, with a template switching oligo incorporated during cDNA synthesis to allow for full length cDNA synthesis and retain strand specificity. Illumina sequencing adapters and barcodes were then added to the cDNA by PCR, followed by cleavage of ribosomal cDNA. Uncleaved fragments were then enriched by PCR for 12-16 cycles. Final library size distribution was again validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen). Indexed libraries were then normalized and pooled for sequencing.