GSM9025795: CITESeq+HTO, progenitor IL7Mut+TLX3; Mus musculus; OTHER
CITE-seq and cell hashing were performed as per the 10X protocol. For every sample, 1 million cells were pelleted and resuspended into 50ul of labelling buffer (PBS +1% BSA). Fc receptors were blocked by adding TruStain FcX (anti-mouse CD16/32, Biolegend, cat. number 101319) to the cell suspension at a concentration of 1ug TruStain FcX per million cells, mixed by gentle pipetting and incubated at 40C for 10 mins. Two pools of antibodies were prepared by combining CD4 and CD8a antibodies with either Hashtag-1 antibody (TotalSeq™-A0301 anti-mouse Hashtag 1, Biolegend, cat. number 155801) (pool1) or hashtag-2 antibody (TotalSeq™-A0302 anti-mouse Hashtag 2, Biolegend, cat. number 155803) (pool2), followed by centrifuging them at 14K rpm at 40C for 10mins. Two individual samples from each group (Thymocyte/IL7-mutant/IL7-TLX-mutant) were labeled either by pool1 or pool2. For labelling, supernatant of the prepared pools was added to corresponding samples to a final concentration of 1ug of individual antibodies/million cells in a total volume of 100ul (volume was maintained using labelling mix). Cells were stained with antibody cocktail for 30 mins at 40C and then washed 3 times with 1.5ml of labelling buffer each. After washing, cells were resuspended in 1 ml of PBS + 0.04% BSA and counted. Approximately 10,000 cells each from two individually labelled samples (pool1/pool2) of each group were then loaded on one channel of the 10X chip (total 20,000 cells loaded per channel) and GEMs (Gel Beads-in-emulsion) were generated using the 10X Genomics Chromium Controller. The passaged cells only included the CITESeq antibodies CD4, CD8, CD11b and B220.The protocol was otherwise unchanged. 3' mRNA-seq gene expression and barcode libraries were then prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3 with Feature Barcoding technology for Cell Surface Protein (10X Genomics) as per the manufacturer guidelines. The barcode libraries were pooled together and sequenced on NextSeq500 as asymmetric paired-end run with a read length of 28 bp for Read 1, 55 bp for Read 2, and 8 bp for the sample index read. The gene expression libraries were pooled and sequenced on a NovaSeq SP (100 cycle) run as asymmetric paired-end run with a read length of 28 bp for Read 1, 80 bp for Read 2, and 8 bp for the sample index read.