GSM8502913: OTI Thy1.1+ CD8+ T in CD11c-GSK3b WT mice 4 days post adoptively transfer, scRNAseq; Mus musculus; RNA-Seq
For isolation of adoptively transferred Pmel-1 Thy1.1+ CD8+ T cells, spleen cells from immunized mice were first enriched with anti-Thy1.1 magnetic microbeads and sorted using BD FACSAira II scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3′ Reagent Kit (v2 Chemistry) and Chromium Single Cell Controller as described in the manufacturer's  instructions. Briefly, FACS-sorted cells were loaded into each reaction for gel bead-in-emulsion (GEM) generation and cell barcoding. Reverse transcription of the GEM (GEM-RT) was performed using a Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems) at 53 °C for 45 min, 85 °C for 5 min, and a 4 °C hold. cDNA amplification was performed after GEM-RT cleanup with Dynabeads MyOne Silane (Thermo Fisher Scientific) using the same thermocycler (98 °C for 3 min, 98 °C for 15 s, 67 °C for 20 s, 72 °C for 1 min, followed by a 12 cycle repeat at 72 °C for 1 min and a 4 °C hold). Amplified cDNA was cleaned up with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA) that was followed by a library construction procedure, including fragmentation, end repair, adapter ligation, and library amplification. An Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) was used for library quality control. Libraries were sequenced on an Illumina HiSeq4000 using a paired-end flow cell: Read 1, 26 cycles; i7 index, 8 cycles; Read 2, 98 cycles.