GSM8061729: T cells from Mtb-infected mixed bone marrow chimera mice, ADT-derived cDNA; Mus musculus; OTHER
FACS isolated cells were pooled and loaded into 2 different lanes on a Chromium Next GEM Chip. Lane 1 was loaded with T cells. Lane 2 was loaded with innate lymphoid cells. Both lanes of the Chromium Next GEM Chip were super-loaded with 25000 cells with a target of 13200 single cells per lane. The scRNA-sequencing libraries were generated using the v3.1 chemistry Chromium Single Cell 3' Reagent Kit (10X Genomics) largely following this kits protocol and the CITE-seq protocol. 0.5 U/µL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was added to single cell RT master mix during the loading step and 1 µL of ADT and HTO additive primers (0.2 µM stock) were added during the cDNA amplification. Following cDNA, ADT, and HTO purification, samples were decontaminated by 2 rounds of centrifugation through 0.2 µM filter microcentrifuge tubes and then removed from the BSL3. Library preparations were completed outside of the BSL3 following the 10X Genomics protocol for the cDNA and the CITE-seq and Cell Hashing Protocol for the ADT and HTO libraries. Quality control of the libraries was performed with a Fragment Analyzer (Agilent). The mRNA, ADT, and HTO libraries were pooled at the following proportions: 50% T cell mRNA, 33% ILC mRNA, 6% T cell ADT, 4% ILC ADT, 4% T cell HTO, and 3% ILC HTO. Libraries were sequenced on a NovaSeq 6000 (Illumina) using one lane of a S1 flow cell and the following cycles read 1 (28 cycles), i7 index (10 cycles), i5 index (10 cycles), read 2 (90 cycles).