GSM6248550: AireKO, mTEClo, input, rep2; Mus musculus; OTHER
Mouse thymus was enzymatically digested and then DAPI-CD45-EpCAM+Ly51-MHCII-high or MHCII-low cells were sorted by FACSAria. 10,000 cells were sorted into 100 uL of BAMBANKER (GC LYMPHOTEC) and kept frozen until library constructions. Cut&tag libraries were constructed as previously reported by Kaya-Okur et al. (CUT&Tag for efficient epigenomic profiling of small samples and single cells, 2019) employing spike-ins for normalizations (Greulich et al., Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation, 2021). Specifically, cell capturing beads for a sample were prepared by incubating 5uL of streptavidin-conjugated magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) with 20ug/mL biotin-conjugated ConA (Sigma-Aldrich, C2272) and 20ug/ml biotin-conjugated WGA (Sigma-Aldrich, L5142) in 100uL of PBS at room temperature for 30min. Then the beads were washed and resuspended in 20uL of binding buffer (20mM HEPES PH7.5, 10mM KCl, 1mM CaCl2, and 1mM MnCl2). Frozen cells in BAMBANKER were thawed and washed with PBS after adding 10,000 293FT cells (Invitrogen) as spike-in. Cells were resuspended in 100uL of wash buffer (20mM HEPES PH7.5, 150mM NaCl, 0.5mM spermidine, and protease inhibitor cocktail; Roche cOmplete ULTRA EDTA-free) and incubated with 20uL of cell capturing beads for 30min. Cells were isolated using a magnetic separator (DynaMag2 Magnet, Invitrogen) and resuspended in 55uL of antibody solution (1:100 diluted anti-H3K27me3 CST#9733 in 20mM HEPES PH7.5, 150mM NaCl, 0.5mM spermidine, protease inhibitor cocktail, 0.05% digitonin; WAKO water-soluble digitonin, 2mM EDTA, and 0.1% BSA). 5uL of the cell suspension was used for preparing the input library as described later and 50uL was incubated overnight at 4C with agitation using a rotary mixer (NISSINRIKA NRC-20D, Tokyo Japan). After the overnight incubation, cells were washed with Dig-wash buffer (0.05% digitonin, 20mM HEPES PH7.5, 150mM NaCl, 0.5mM spermidine, and protease inhibitor cocktail) and incubated in the secondary antibody buffer (1:100 diluted anti-rabbit IgG; ROCKLAND 611-201-122 in Dig-wash buffer) at 4C for 1hr with agitation. After the incubation, cells were washed 3 times with Dig-wash buffer and incubated in 100uL of 8ug/mL in-house pAG-Tn5 in Dig-300 buffer (20mM HEPES PH7.5, 300mM NaCl, 0.5mM spermidine, 0.01% digitonin, and protease inhibitor cocktail) at room temperature for 1 hour with agitation. In-house pAG-Tn5, in which Protein A and Protein G IgG binding domains were fused to Tn5 transposase, was prepared by utilizing pTXB1-Tn5 employing regular methods in molecular biology following the previous reports (Picelli et al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects, 2014 and Hennig et al., Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol, 2018. pTXB1-Tn5 was a gift from Rickard Sandberg. Addgene plasmid #60240; http://n2t.net/addgene:60240). Then, cells were washed 3 times with Dig-300 buffer, resuspended in 100uL of Tagmentation buffer (Dig-300 buffer containing 10mM MaCl2), and incubated at 37C for 1hr mixing at 600rpm (ThermoMixer, Eppendorf) for DNA Tagmentation. The reaction was stopped by adding EDTA, SDS, and DNase-free RNaseA to 15mM, 0.5%, and 0.05mg/mL respectively, and incubated at 37C for 15min mixing at 600rpm. Then, tagmented DNA was purified by proteinase K treatment (Proteinase K, recombinant, PCR Grade, Roche) followed by phenol-chloroform extraction. DNA was amplified using indexed primers and KAPA HiFi DNA Polymerase (Roche) to construct an NGS library. The libraries were purified using AMPure XP beads (x1.1 vol) and quantified by MultiNA (SHIMADZU). For preparing the input libraries, DNA was purified from 5uL of cell suspension by proteinase K treatment followed by phenol-chloroform extraction. DNA tagmentation was performed using an in-house Tn5 enzyme prepared as previously described (Picelli et al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects, 2014 and Hennig et al., Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol, 2018. pTXB1-Tn5 was a gift from Rickard Sandberg. Addgene plasmid #60240; http://n2t.net/addgene:60240). Then, Tn5 was dissociated by adding SDS to 0.04% and DNA was amplified by PCR using indexed primers and KAPA HiFi DNA Polymerase. The libraries were purified and size-selected by using AMPure XP beads (x0.55 vol. to remove large fragments and then x1.0 vol. for omitting smaller fragments). Libraries were quantified by MultiNA and pooled for next-generation sequencing.