Project description:Purpose: to identify the binding sites for BATF2 in HSCs during M. avium infection, we conducted CUT&RUN sequencing of the HSCs from infected WT mice using an anti-BATF2 antibody. We used an anti-IgG antibody as a control to exclude noise and nonspecific binding and an anti-H3K27ac to assess open chromatin Methods: 10,000-50,000 HSCs (CD45.1/CD45.2 KL CD150+ CD48-) into HBSS from1-month infected WT mice (n=10-12 per group). CUT&RUN sequencing was performed with modified methods as previously described (Henikoff et al., 2020; Skene et al., 2018). 50,000 HSCs (LK CD150+, CD48-) were sorted pools of naïve or 1-month (M. avium) infected WT mice for CUT&RUN sequencing (N=7-8). Cells were washed with Wash Buffer (50mL total with 20 mM HEPES pH 7.5, 150mM NaCl, 0.5 mM Spermidine, and one Roche Complete protein inhibitor tablet) and bound to Concanavalin A-coated magnetic beads (Bang Laboratories L200731C) for 15 minutes room temperature. Sample slurries were magnetically separated, washed, and incubated with primary antibody diluted in wash buffer containing 0.05% digitonin (Dig Wash) overnight at 4 °C. Slurries were then magnetically separated and washed with Dig Wash three times and then incubated with protein A-MNase (pA-MN, Epicypher 15-1016) for 1 hour at 4 °C. Slurries were magnetically separated and washed, resuspended in 200uL Dig Wash, and placed on an iced heating block. 2uL of 2mM CaCl2 was added to catalyze the pA-MN reaction and digestion. After 45 minutes, one equal volume of Stop Buffer (340mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/mL glycogen, 5 ug/mL RNase A, 2 pg/mL heterologous spike-in DNA) was added to end the reaction, and sample slurries were then incubated for 30 minutes at 37 °C to release fragments and then magnetically separated. The supernatant was collected, and DNA was purified via phenol-chloroform extraction and ethanol precipitation. Samples were resuspended in molecular-grade water after ethanol precipitation. DNA was quantified with Qubit 2.0 DNA HS Assay (ThermoFisher, Massachusetts, USA) and quality was assessed by Tapestation High sensitivity D1000 DNA Assay (Agilent Technologies, California, USA). Library preparation was performed using the KAPA Hyper Prep kit (Roche, Basel, Switzerland) according to the manufacturer’s recommendations. Library quality and quantity were assessed with Qubit 2.0 DNA HS Assay (ThermoFisher, Massachusetts, USA), Tapestation High Sensitivity D1000 Assay (Agilent Technologies, California, USA), and QuantStudio ® 5 System (Applied Biosystems, California, USA). Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq (Illumina, California, USA) with a read length configuration of 150 paired-end (PE). Antibodies used were anti-BATF2, Rabbit-anti mouse IgG (Jackson ImmunoResearch 315-005-003), and Rabbit anti-H3K27ac (Cell Signaling Technologies 8173T). PE reads were aligned to mm10 using Bowtie2 version 2.4.5. MACS2 version 2.2.7.1 was used to call peaks and CUT&RUN IgG was used as a negative control. Results: We found binding of BATF2 at cis-elements of ccr5, ifngr2 (interferon gamma receptor 2), ly6c1, and ccl28 genes (Figure 7C). The locations were highly aligned with accessible chromatin positions marked by H3K27ac, suggesting that BATF2 enhanced gene expression in response to infection. The observations were aligned with our RNA-seq analysis of gene expression in WT or Batf2 KO HSCs in the presence or absence of M. avium infection, as ccr5, ifngr2, ly6c1, and ccl28 were more effectively induced in WT compared to Batf2 HSCs during M. avium infection (Figure S5B). Conclusion: BATF2 interacts with regulatory elements in IFN response genes and facilitates increased expression in response to infection
2023-01-19 | GSE206276 | GEO