Anti h3k9me2

We thawed the cortices derived from a single brain and filtered the tissues into a single-cell suspension using gentle mechanical dissociation. We washed cells twice with PBS containing protease inhibitor cocktail (Cat# 78437; Thermo Scientific) and re-suspended them in medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [33 (link)]. We carried out chromatin immunoprecipitation reactions following the previously published protocol [34 (link)]. The specific antibodies used in this study include anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), antiH3K9me3 (Cat# 05-1242; Millipore-Sigma, RRID:AB_1587136), antiH4K20me3 (Cat# 91107; Active Motif, RRID:AB_2793777), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and the negative IgG control (Cat# SC-2027; Santa Cruz, RRID:AB_737197). We used antibodies for modified histones and the IgG control at a concentration of 1 µg/ChIP reaction and 4 µg/ChIP reaction for SATB2. We purified precipitated DNA using the QIAquick PCR Purification Kit (catalog # 28106, Qiagen) and assayed the enrichment of the indicated sequences using quantitative PCR. We performed qPCR using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 1.

We isolated protein from the GD17 fetal cortex using the AllPrep DNA/RNA/Protein Mini Kit (Catalog No. 80004; Qiagen) according to the manufacturer’s instructions. We then separated 5–10 µg of protein on 10–15% sodium dodecyl sulfate-polyacrylamide gels and transferred proteins to nitrocellulose membranes. The primary antibodies we used in this study are: anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), anti-pan histone H3 (Cat# ab1791; Abcam, RRID:AB_302613), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and anti-Tubulin (Cat# ab40742; Abcam, RRID:AB_880625). We visualized blots using secondary antibodies conjugated to horseradish peroxidase (catalog# sc-2004; RRID:AB_631746; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). We calculated relative levels of H3K9me2 as a ratio to total histone H3, and relative levels of SATB2 as a ratio to Tubulin. We quantified band intensities by densitometry using ImageJ (RRID:SCR_003070; National Institutes of Health, Bethesda, MD, USA). Each experimental group contains protein extracts derived from 5 control, 3 unaffected, 4 affected animals.

Cells were grown to 80 % confluence, washed twice in warm PBS, dissociated using Accutase (Cat# SCR005; Millipore), and resuspended in warm medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [89 (link)]. ChIP reactions were performed as described previously [35 (link)] followed by DNA purification using a Qiaquick PCR Cleanup kit (Cat# 28106; QIAGEN). Antibodies used include: anti-H3K4me3 (Cat# 04-745; Millipore), anti-H3K27me3 (Cat# 39155; Active Motif), anti-H3K9ac (Cat# 07-352; Millipore), and anti-H3K9me2 (Cat# 39239; Active Motif). Antibodies for modified histones were used at 1 µg/ChIP reaction. The concentration of IgG (Cat# SC-2027; Santa Cruz) was also used at 1 µg/ChIP. For analysis of candidate loci, real-time PCR was performed using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) according to the recommended protocol. Reactions were performed on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 3.

After stimulation with anti-IgM (10 µg/ml) or LPS (2 µg/ml) for 24 h or 48 h, splenic B cells were cross-linked with 1% fresh formaldehyde for 10 min at room temperature, neutralized with glycine for 5 min and lysed in SDS lysis buffer. The cross-linked DNA was then sonicated and sheared into fragments 200–1000 base pairs in length with UCD-300 (Bioruptor). ChIP experiments were performed using a Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions to obtain ChIP-enriched DNA. For the ChIP-Seq assay, the 75-nt sequence reads generated by Illumina sequencing were mapped to the genome using the Burrows-Wheeler Aligner algorithm with default settings. Only reads that passed Illuminas purity filter, aligned with no >2 mismatches, and mapped uniquely to the genome were used in the subsequent analysis. Average of peak values of all active regions in the gene and within the gene margin were used to calculate the differentially enriched genes. For the ChIP-qPCR assay, subsequent qRT-PCR was performed to quantify the ChIP-enriched DNA. The data were normalized to the input. The antibodies for ChIP, including anti-H3K9me2 (39239, 1:100, 10 μg per ChIP), anti-H3K9me3 (39161, 1:100, 10 μg per ChIP) were purchased from Active Motif. The primers used for ChIP-qRT-PCR are listed in Supplementary Table 2.