GSM7026710: HSPCs, HTO-derived cDNA; Mus musculus; OTHER
Lineage-Sca1+cKit+ cells were FACS-purified, counted and checked for viability using hematocytometer. Cell suspension was adjusted to a concentration of 1,250 cells/µL and 54,000 cells were loaded onto Chromium Controller (10x Genomics). Libraries were generated using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics). mRNA and hashing oligos (HTO) were reverse transcribed, barcoded and purified using a Dynabeads bead cleanup mix. The cDNA was amplified by PCR according to the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 protocol with additional primer amplifying the HTO-derived tags (5'-GTGACTGGAGTTCAGACGTGTGCTC-3'). SPRI size selection was performed to separate fragments containing mRNA-derived (>300 bp) and HTO-derived (<180 bp) cDNA. mRNA-derived cDNA sequencing library was next generated according to manufacturer's instructions (10x Genomics). HTO-derived sequencing library was prepared according to CITE-seq_and_Hashing_protocol_190213 (https://cite-seq.com/protocols). HTO-derived fragments we purified with 2X SPRI beads (Beckman Coulter) and amplified using the KAPA HiFI HotStart Library Amplification Kit (Roche) with SI-PCR primer (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3') as i5 primer (Illumina), TruSeq D708_s primer 5'-CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTGGAGTTCAGACGTGTGC-3' as i7 primer (Illumina) and the following cycling conditions: 95°C for 3 min followed by 12 cycles 95°C for 20 sec; 64°C for 30 sec; 72°C for 20 sec and final elongation 72°C for 5 min. Final cDNA- and HTO-derived libraries were pooled and sequenced on Illumina NovaSeq 6000 instrument using S2 Reagent Kit v1.5 (100 cycles). Preparation of libraries and sequencing were done at the Center for Translational Genomics, Lund University.