GSM4905101: WT CD4+GFP+ Treg cells; Mus musculus; RNA-Seq
After euthanasia, spleens from WT and CD25-Y129H Foxp3-GFP mice were collected. Single cell suspensions were obtained by filtering through nylon gaze and re-suspended in FACS buffer. Treg cells were sorted based on CD4 and GFP expression. Cells from three mice per genotype were labelled with TotalSeq cell hashing antibodies (Biolegend), mixed together, and used to generate single-cell gel-beads in emulsion using Chromium Single Cell 3' Library & Gel Bead Kit v.3 according to the manufacturer´s protocol (10x Genomics). The scRNA-seq libraries were prepared following the user guide manual (CG000183 Rev A) provided by the 10X Genomics and Chromium™ Single Cell 3' Reagent Kit (v3). Briefly, after labeling with cell hashing antibodies, 9000 cells from each sample were encapsulated in microfluidic droplets (estimated doublet rate ~4.3%). Encapsulated cells were subjected to reverse transcription (RT) reaction at 53ºC for 45 min. After RT step, the emulsion droplets were broken and barcoded-cDNA and -HTO was purified with DynaBeads, followed by 12-cycles of PCR-amplification (98 °C for 180 s; [98 °C for 15 s, 63 °C for 20 s, 72 °C for 60 s] x 12-cycles; 72 °C for 60 s). Afterwards, PCR products were cleaned using SPRI selection and in this step HTO-libraries were separated from cDNA libraries as descibed in cell hashing protocol (version 2019-02-13, https://cite-seq.com/protocol/; PMID: 30567574). For cDNA libraries, 10 µl of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of ~400 bp. Next, the DNA library was ligated to the sequencing adapter followed by indexing PCR (98 °C for 45 s; [98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s] x 16 cycles; 72 °C for 60 s). The resulting DNA library was double-size purified (0.6-0.8X) with SPRI beads. For HTO libraries, they were amplified and ligated to sequencing adaptors using 2x KAPA Hifi PCR (95 °C for 45 s; [95 °C for 20 s, 64 °C for 30 s, 72 °C for 20 s] x 12 cycles; 72 °C for 300 s) and purified with SPRI beads (1.6x). Both cDNA- and HTO-libraries were sequenced on an Illumina NextSeq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 56 cycles) resulting in ~300 million reads per cDNA sample (average reads per single-cell being 100,000 a).