CUT&RUN sequencing on wildtype HSCs, MPP3s, GMPs, and macrophages with four weeks M. avium infection

Purpose: Central trained immunity is a phenomenom in which reprogrammed HSPCs differentiate into innate immune cells with heightened immune responses. To determine whether histone modifications are conserved and passed down to downstream lineage cells, we performed CUT&RUN sequencing on various HSPC subpopulations and macrophages in both M. avium infected and naive states. This was done to determine if there were conserved changes across all subpopulations, which would imply to us if specific gene signatures and genomic loci could be conserved for eliciting central trained immunity. Overall design: CUT&RUN-sequencing protocol was performed with slight modifications previously published work from the lab and others (Skene and Henikoff et al., Elife 2017 and Le et al., iScience 2023). Bone marrow from M. avium infected or sham-treated mice was isolated from tibias, femurs, and pelvic bones and pooled per group. After red blood cell lysis, cells were separated by cKit-enrichment and stained for specific cell surface markers. From cKit+ postivie fractions, 30,000 HSCs (Lin-,cKit-enriched, CD150+, CD48-), 200,000 MPP3s (Lin-, cKit-enriched, CD150-+, CD48+, Flk2-, CD34+), 200,000 GMPs (Lin-, cKit-enriched) were sorted per sample. 200,000 macrophages (CD3-, B220-, NK1.1-, CD11b+, Ly6g-, Ly6c+) were sorted per sample from the cKit- fractions. Samples were counted and washed with wash buffer (50 mL H2O with 20 mM HEPES (GIBCO 15630080) three times. Cells were then incubated with activated concanavalin a beads (Bang Laboratories L2007231C) for fifteen minutes at room temperature. Sample slurries were then washed one more time and resuspended in wash buffer supplemented with 0.5% Digitonin (Dig-wash buffer). Dig-wash buffer was used for the remainder of the experimental method, with samples and buffers were kept on ice or at 4°C. Samples were then incubated with histone modification antibodies overnight at 4°C on a rotator. Depending on the sample, the antibodies used were targeted for Rabbit anti-H3K27ac (Cell Signaling Technologies 8173, 1:50 concentration), Rabbit anti-H3K427me3 (Cell Signaling Technologies 9733, 1:50 concentration), and Rabbit-antimouse IgG (Jackson ImmunoResearch 315-005-003, 1:50 concentration) for negative controls. Samples were washed once again three times and incubated with pAG-MNase (Epicypher SKU:15-016 ) on a rotator for one hour in 4°C. After, samples were washed three times and then resuspended in 150uL Dig-wash buffer and 2uL of 100mM CaCl2 was added to catalyze MNase activity and cutting. Cell-bead homogenates were kept in 4°C for 40 minutes while the reaction occured. The reaction was quenched with 2X stopping buffer (5 mL total, H2O, NaCl 340mM, EDTA 20 mM, EGTA, 4 mM, Digitonin 0.05%, RNAse A 100ug/mL, Glycogen 50 ug/mL, and Spike-in DNA 1ng/mL), and samples were heated to 37 C for 20 minutes for MNase/Histone/DNA complex release from cells and out into the supernatant. Samples were placed on magnetic stands to separate the DNA supernatant from the bead slurry, and DNA purification was completed via phenol chloroform and ethanol precipitation extraction. Libraries were generated with the services of Admerahealth. Quantified DNA by Qubit 2.0 HS DNA (ThermoFisher, Massachusetts, USA) was assessed by Tapestation High on a D1000 High sensitivity DNA Assay (Agilent Technologies, California, USA). Library preparation was completed using the KAPA HyperPrep kit (Roche, Basel, Switzerland) per the manufacturer's instructions. Library quality was assessed with Qubit 2.0 (ThermoFisher, Massachusetts, USA), Tapestation (Agilent Technologies, California, USA), and QuantStudio 5 System (Applied Biosystems, California, USA). Illumina 8-nt dual indices were used. Libraries were pooled equimolarly based on quality and sequenced on an Illumina NovaSeq (Illumina, California, USA) with a 150 paired-end (PE) read length.