description: Protocols: All mice were housed under specific-pathogen free conditions and euthanized by approved schedule 1 methods following local guidelines. Single cell suspensions were generated from tissues and incubated with antibody cocktails in FACS buffer (PBS with 2% FCS and 2 mM EDTA) for 15 min at 4°C, then specific populations of interest obtained by sorting cells on a BD Aria cell sorter. Dead cells were excluded by use of a Live/Dead Fixable dye (Biolegend). HSPCs were identified as follows: LSK+MyP: CD45+Lin (CD3ε, TCR-β, CD19, B220, NK1.1, Ter119, Siglec F, Ly6G, FcεR1α, CD11b, CD11c, Ly6C)-Sca-1+/-cKit+. Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol. Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol. Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16- nucleotide 10x Barcode, a 12-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs then yielded barcoded cDNA from poly-adenylated mRNA. Following incubation, GEMs were broken and pooled fractions recovered. First-strand cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5 & P7 sequences, a sample index, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.
attributes.tag: ENA first public
attributes.value: 2021-03-01
attributes.tag: ENA last update
attributes.value: 2021-01-27
attributes.tag: External Id
attributes.value: SAMEA7875238
attributes.tag: INSDC center alias
attributes.value: UNIVERSITY OF MANCHESTER
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF MANCHESTER
attributes.tag: INSDC first public
attributes.value: 2021-03-01T12:41:39Z
attributes.tag: INSDC last update
attributes.value: 2021-01-27T16:56:52Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10050:BM
attributes.tag: age
attributes.value: 8 to 16
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: hematopoietic cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: 1
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-10050:BM
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6