title: GSM4419004: WT rep2; Mus musculus; RNA-Seq
library_construction_protocol: CD8+ TILs were isolated from MC38 tumor-bearing Cd4-Cre+Lsd1f/f and Lsd1f/f mice on day 18 as described above. CD8+ TILs from two individual tumor-carrying Lsd1f/f mice were combined as a biological replicate, and CD8+ TILs from each individual tumor-carrying Cd4-Cre+Lsd1f/f mouse was used as a biological replicate. CD8+ TILs were stained with antibodies against CD45.2 (clone 104), TCR (clone H57-587), CD8 (clone YTS156.7.7), CD44 (clone IM7) and PD-1 (clone 29F.1A12), and 7-AAD Viability Staining Solution was used to exclude dead cells. The CD45.2+TCR+CD8+CD44+PD-1+ cells were then sorted on a BD Aria. Approximately 10,000~20,000 sorted cells for each biological replicate were directly lysed in 1 ml TRIzol Reagent (Life Technologies, cat#15596018). 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 (Qiagen, cat#74004) 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 rRNA-depleted RNA with a NEBNext® rRNA Depletion Kit (New England Biolabs, cat#E6310S) according to the manufacturer's instructions. The rRNA-depleted RNA was purified with a RNA Clean & Concentrator-5 kit (Zymo Research, cat#R1016) and then used to generate a directional RNA library with a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, cat#E7760L) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, cat#E7335L) according to the manufacturer's instructions. Library concentrations and quality were assessed on a Bioanalyzer and by qPCR. The library was sequenced at Nextseq 500 (Illumina) to generate reads from paired-ends (43 bp + 42 bp).
attributes.tag: GEO Accession
attributes.value: GSM4419004