GSM4910282: Lsd1 KO rep2; Mus musculus; RNA-Seq
CD8+ TILs were isolated from B16 tumor-bearing wild-type mice on day 14. Two individual tumors were combined as one biological replicate. CD8+ TILs were stained with antibodies against CD45.2, TCRbeta, CD8. 7-AAD Viability Staining Solution was used to exclude dead cells. The CD45.2+TCRbeta+CD8+ cells were then sorted on a BD Aria. Approximately 30,000~70,000 sorted cells for each biological replicate were directly lysed in 1 ml TRIzol Reagent. After incubation for 5 mins, 0.2 ml chloroform was added and mixed by inverting the tubes several times. Samples were incubated for 2~3 mins and later centrifuged for 15 minutes at 12,000g at 4 °C. The upper aqueous phase containing the RNA was collected and mixed with equal volume of 70% ethanol, which was then loaded into a spin column from a RNeasy Micro Kit and subjected to RNA isolation according to the instruction manual. The on-column DNase digestion was conducted to eliminate DNA contamination. Purified total RNA was quantified by Qubit (Invitrogen) and used to generate polyA+ enriched RNA. The purified polyA+ RNA was used to generate a directional RNA library with a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®  and NEBNext® Multiplex Oligos for Illumina® according to the manufacturer's instructions. Library concentrations and quality were assessed on a Bioanalyzer and by qPCR. The library was sequenced at Novaseq (Illumina) to generate reads from paired-ends (2X150bp).