GSM6327094: Treg, TCR losing, 21 days, rep1 [H28_neg]; Mus musculus; RNA-Seq
Cells sorted directly into Trizol LS; phase separation was induced with 200 µL chloroform. RNA was extracted from 350 µL of the aqueous phase using the miRNeasy Micro Kit (Qiagen catalog # 217084) on the QIAcube Connect (Qiagen) according to the manufacturer's protocol. Samples were eluted in 13 µL RNase-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 122-400pg total RNA with RNA integrity numbers ranging from undetectable to 9 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. Subsequently, 4ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run with the NovaSeq 6000 S4 Reagent Kit (Cycles), (Illumina).