GSM6032028: CMC_day7_mouse2_plate1 [L7-CMC-M2P1i7]; Mus musculus; RNA-Seq
At day 4 post Lm-N4 infection, splenocytes were enriched for CD45.1+ OT-1 T-cells using anti-CD45.1 biotin/ anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech, Bergisch-Gladbach, Germany). Single-cells were sorted on BD FACS Fusion (100 micron nozzle, standard operation settings, single-cell purity, index sorting). Individual cells meeting the gating strategy were sorted directly in lysis buffer into individual wells of a low-binding PCR plate. Immediately after sorting, each plate was spun down, snap-frozen on dry ice and stored at -80oC until use. The single-cell libraries were generated using the previously described SCRB-seq protocol, with some modifications. After RNA purification and reverse transcription, the generated single-cell cDNA was amplified for 20 cycles. The barcoded single-cell amplicons were double-purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA was used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024, Illumina). After PCR amplification of the fragmented libraries, the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 µl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626, Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010, Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600, Kapa Biosystems).