GSM8483323: C57BL/6J-ΔEPE3, thymocyte, CD4(-)/CD8(-), replicate2 [pk3-2_DN-2]; Mus musculus; RNA-Seq
Thymocytes were depleted using CD8α microbeads. The CD8-depleted fraction was incubated with lineage marking antibodies (TCRβ, CD4, and CD8, CD11c, CD11b, Gr1, CD19, B220, Ter119) and then lineage- cells were sorted in RPMI-1640 medium supplemented with 10% FBS. Post-sorting, a small fraction of sorted cells was used to check the purity (>98%). 200,000 cells per sample were provided for the library preparation. Using the Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1  and the Chromium Next GEM Chip H Single Cell Kit v1.1 , thymocyte nuclei, at 7,000 nuclei per µl, were loaded onto a Chromium controller  to generate single-nuclei GEMs after which library construction is performed according to the manufacturer's protocol Approximately 10,000 cells per sample were added to chip G to create GEMs. Cells were lysed, and the bead captured poly(A) RNA was barcoded during reverse transcription in a thermal cycler at 53 °C for 45 min, followed by 85 °C for 5 min. cDNA was generated and amplified. Quality control and quantification of the cDNA were conducted using Agilent's High Sensitivity DNA Kit in the 2100 Bioanalyzer. scRNA-seq libraries were constructed using a Chromium Single Cell 3ʹ Library Kit v3.1 and indexed with Single Index Kit T Set A. The libraries were sequenced using an Illumina NovaSeq 6000 sequencer with single-indexing strategy consisting of 28 cycles for read 1 and 91 cycles for read 2.