GSM4494904: HSPCs, HTO-derived cDNA; Mus musculus; OTHER
Lin-Sca1+ckit+ (LSK) cells from two WT and two Helios deficient mice were purified by flow cytometry. Purified cells were then stained with Antibody Derived Tag directed against CD41, CD48, CD150 and Flt3 and barcoded with a unique Oligonucleotide Hash Tag. After Hash- and Cite-tagged cells were counted and checked for viability using a hematocytometer, 10e4 cells (2500 LSK cells for each mouse) were loaded into a Chromium Controller (10X Genomics). Library was prepared using Chromium Single Cell 3ʹ v2 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3' v2 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer's instructions for Single Cell 3' v2 protocol (10x Genomics). For antibody-derived tagged library, we used the KAPA HiFI HotStart Library Amplification Kit (Roche) with the following primers and amplification program: -CITE-seq library: amplification was performed at 95°C for 3' followed by ten cycles at 95°C f¬¬or 20''; 60°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' Small RNA RPI1 primer as i7 primer (Illumina). -HASH-seq library: amplification was performed at 95°C for 3' followed by ten cycles 95°C for 20''; 64°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC-3' TruSeq D701_s primer as i7 primer (Illumina). The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3' SI-PCR primer as i5 primer (Illumina) was used for both CITE- and HASH- seq library amplification. Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% HTO library before sequencing.
GEO
GSM4494904
GEO Accession
GSM4494904