title: GSM8899159: H3K9me3 CUT&RUN in iGMPs Replicate 1; Mus musculus; OTHER
library_name: GSM8899159
library_construction_protocol: H3K4me1 and H3K9me3 CUT&RUN was performed as previously described in uninduced iGMPs using the CUTANA ChIC/CUT&RUN Kit (14-1048, EpiCypher; Franklin et al. 2022; Skene et al. 2018). Briefly, 1 million cells were collected for each sample. Cells were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor) and bound to concavalin A beads (21-1401, EpiCypher), and incubated overnight at 4 °C with 1:100 or 1:50 (IgG) dilution of primary antibody (Wash Buffer, 0.01% digitonin, 2 mM EDTA). The following antibodies were used: H3K4me1 (ab8895, Abcam), H3K9me3 (ab8898, Abcam), and IgG (13-0042, EpiCypher). Cells were then washed twice with wash buffer. pAG-MNase (15–1116, EpiCypher) was then used at 1:20 dilution for each 50 μL CUT&RUN reaction and incubated for 10 min at room temperature. Cells were then washed twice with wash buffer and 2 mM CaCl2 was added for 2 hours at 4 °C. Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen) was added at 1.5x dilution, with 0.5 ng E. coli spik-in DNA per reaction, and incubated for 10 min at 37 °C. DNA was purified with CUTANA DNA purification kit (14-0050, EpiCypher). NEB Ultra II DNA Library Prep and Index Kits (E7645, E6440) were used according to the manufacturer's protocol with modifications. Briefly, up to 10 ng DNA was used for end preparation and adapter ligation without size selection. NEB indexes were added with 2-step PCR using an optimized number of cycles (12-14) and short extension times (10 s). Fragment distribution of prepared libraries were confirmed by Bioanalyzer to contain nucleosomal and sub-nucleosomal enrichment and then submitted to Illumina Novaseq 6000 PE100 sequencing at the University of California Irvine Genomics Research and Technology Hub.
identifiers.namespace: GEO
identifiers.id: GSM8899159_r1