GSM7951438: Library_1; Homo sapiens; RNA-Seq
Cells were trypsinized Samples were then stained for multiplexing using cell hashing, using the Cell Hashing Total-Seq-ATM protocol (Biolegend) following the protocol provided by the supplier, using 4 distinct Hash Tag Oligonucleotides-conjugated mAbs. Briefly, for each condition, 1.106 cells were resuspended in 100µL of PBS, 2% BSA, 0.01% Tween and incubated with 10µL Fc Blocking reagent for 10 minutes at 4°C then stained with 0.5µg of cell hashing antibody for 20 minutes at 4°C. After washing with PBS, 2% BSA, 0.01% Tween, samples were counted and merged at the same proportion, spun 5 minutes 350 x g at 4°C and resuspended in PBS supplemented with 0.04% of bovine serum albumin at final concentration of 500 cells/µL. Samples were then adjusted to the same concentration, mixed in PBS supplemented with 0.04% of bovine serum albumin at a final concentration of 100 cells/µl and pooled sample were immediately loaded onto10X Genomics Chromium device to perform the single cell capture. After single-cell capture on the 10X Genomics Chromium device (3' V3), libraries were prepared as recommended, following the Chromium Next GEM Single Cell  3' Reagent  Feature Barcoding V3.1 kit (10X Genomics) and a targeted gRNA amplification with respectively 6, 8 and 10 PCR cycles. Libraries were then quantified, pooled (80% RNA libraries, 10% gRNA libraries and 10% hashing libraries) and sequenced on an Illumina NextSeq 2000.