Hiseq 4000 machine

Library preparation for RNA sequencing was carried out using TruSeq RNA Library Prep Kit v2 (Illumina) according to the manufacturer's instructions. Libraries were sequenced using paired‐end sequencing length of a 100 nucleotides on a HiSeq 4000 machine (Illumina).

Viable cells were counted with Trypan Blue (Thermo Fischer Scientific, Cat#T8154) solution, and for each cell line, 1,000–2,000 viable cells were loaded into a chip and processed with the Chromium Single Cell Controller (10X Genomics, Pleasanton, California United States, estimated recovery: 1,500 cells/sample). Single-cell RNA-Seq libraries were prepared using the Single Cell 3′ GEM, Library and Gel Bead Kit V3 (10× Genomics, Cat#PN-1000075) according to manufacturer instructions. Briefly, single cells were partitioned into individual Gel Beads-in-emulsion (GEMs) and the RNA obtained from lysed cells was barcoded through reverse transcription. Each cell is encapsulated in a gel bead that contains a unique 14- base pair (bp) molecular barcode, a 10-bp randomer to index molecules (unique molecular identifier, UMI), and an anchored 30-bp oligo-dT to prime polyadenylated RNA transcripts. DynaBeads® MyOne Silane Beads (Thermo Fischer Scientific, Cat#37002D) were used to purify the resulting barcoded cDNA that was subsequently amplified via PCR (12-14 cycles, according to the available cDNA quantity). Libraries were then checked and quantified via the Agilent 2100 Expert Software. The resulting libraries were sequenced across one lane using an Illumina Hiseq4000 machine (Supplementary Table S12).

Chromatin immunoprecipitation was performed on murine Lin⁻ BM HSPCs as previously described (Horton et al., 2017 (link)). Cells were cross-linked with 1% formaldehyde for 5 min for histone markers (H3K27me3, H3K27Ac, and H3K4me3; Millipore, Abcam, and Diagenode, respectively). ChIP-seq library preparation of ChIP DNA or input DNA was performed using TruSeq ChIP Sample Prep Kit (Illumina). KAPA Library Quantification kit (Kapa Biosystems) was used for library DNA quantification. Average library size was determined using an Agilent DNA 1000 Kit (Agilent Technologies) run on a 2100 Bioanalyzer System (Agilent Technologies). Libraries were then pooled for multiplexing for single-read sequencing on an Illumina HiSeq 4000 machine at the Genomics Core, CRUK Cambridge Institute. Experiments were performed in duplicate on biologically independent samples.

The genomic DNA was obtained from overnight cultures of the isolates in LB using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). The quality of gDNA samples was assessed using the Qubit dsDNA BR Assay Kit on a Qubit 2.0 device (Thermo Fisher Scientific) and by running the samples on 1% agarose gels with Midori Green (Nippon Genetics Co) staining to assess potential shearing. The gDNA was subsequently prepared for Illumina sequencing using either a TruSeq DNA PCR-Free kit (Illumina) or the NEBNext Ultra DNA Library Prep kit (New England Biolabs) and sequenced on an HiSeq 4000 machine (Illumina) using a paired-end approach (2*151 bp). Wild-type strains gDNA were also sequenced by long-read PacBio RSII technology to fully resolve the chromosome.