GSM6626484: Unstimulated_0_hrs_Don1; Mus musculus; RNA-Seq
For mRNA sequencing, cells were stained for viability, CD8 expression and CD103 expression. Viable cells were sorted for CD8+ and CD103-. Sorted cells were lysed in 200 µl TRIzol (Thermo Fisher, 15596026) and stored at -80°C before sending to Single Cell Discoveries B.V. Sequencing was performed using an adapted version of the CEL-seq protocol. In brief: Total RNA was extracted using the standard TRIzol protocol and used for library preparation and sequencing. mRNA was processed as described previously, following an adapted version of the single-cell mRNA seq protocol of CEL-Seq.[42,43] In brief, samples were barcoded with CEL-seq primers during a reverse transcription and pooled after second strand synthesis. The resulting cDNA was amplified with an overnight in vitro transcription reaction. From this amplified RNA, sequencing libraries were prepared with Illumina Truseq small RNA primers. The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles).