GSM4971293: KO_HSC_H3.3_rep_1; Mus musculus; ChIP-Seq
HSCs were sorted into PBS + 0.1 mg/ml BSA in DNA LoBind tubes and centrifuged at 2000 rpm for 5 minutes in a swing bucket rotor directly followed by centrifugation in a table-top centrifuge at 7000 rpm for 1 minute. The supernatant was discarded before adding 100 μl TM2 buffer (10 mM Tris [pH 7.4], 2 mM MgCl2, 1 mM CaCl2, 0.6% NP-40) + 1:100 protease inhibitor (Calbiotech) to lyse cells and pipet gently to mix. Samples were incubated on ice for 4 minutes before adding micrococcal nuclease (MNase) depending on the cell number (25 GU for 5K-10K cells = 5 μl of 1:400 MNase diluted in TM2 buffer). Samples were then incubated at 37oC for 2 minutes before adding 100 μl 2xRIPA (20 mM Tris [pH 7.5], 280 mM NaCl, 1.8% Triton X-100, 0.2% SDS, 0.2% Sodium deoxycholate, 5 mM EDTA) + 1:100 protease inhibitor (Calbiotech) + 5 μl 0.5M EDTA and centrifuging at 13,200 rpm 15 minutes. Samples were transferred to a 200 μl PCR tube and 20 μl (10%) were taken for input before adding 1 μl antibodies to the remaining 180 μl of sample for IP (anti-H3.3 [MABE872, rat mAb]). Tubes were then rotated at 4oC overnight (12-16 hours). 10 μl of protein-G Dynabeads were washed with 500 μl of 1X RIPA twice in a 1 ml tube and reconstituted by adding 10 μl of 1X RIPA per IP sample. The IP samples were centrifuged briefly to remove liquid on the lid before adding 10 μl of washed Dynabeads to each IP sample. IP samples were then rotated at 4 oC for 2 hours before being centrifuged briefly to remove liquid from caps. Tubes were then placed on a magnetic rack to collect beads before removing and keeping the supernatant to use for an MNase digestion check. Beads were then resuspended in 200 μl 1X RIPA before the beads were again collected on a magnet and the supernatant discarded. This RIPA wash was repeated for a total of 2 RIPA washes. Two LiCl buffer washes were performed in the same way. The beads were then resuspended in 200 μl TE buffer and transferred to a new PCR tube (tube switch). The tubes were placed on a magnet to collect the beads before the TE buffer was discarded. 40 μl TE buffer + 2 μl Proteinase K (10 mg/ml) were then added to IP samples. 20 μl TE buffer + 2 μl Proteinase K (10 mg/ml) were added to input samples. Tubes were incubated at 37 oC for 1 hour. The IP (immunoprecipitated), input, and supernatant DNA was then purified with the ZYMO DNA clean and concentrator kit (1:5 binding buffer). The IP and input DNA were eluted in 40 μl TE buffer. The supernatant DNA was eluted in 10 μl TE buffer and the MNase digestion efficiency was checked on a 5% PAGE gel. A 2.0x SPRI cleanup was then performed and the beads were briefly air dried before resuspension in 5 μl of elution buffer and mixing (did not elute—kept beads through the adaptor ligation reaction). The NEBNext Ultra II kit was used to generate libraries for Illumina sequencing using adaptors diluted to 1:10 for input samples and 1:25 for IP samples. A 1.3x SPRI cleanup was performed after adaptor ligation and DNA was eluted in 17 μl of elution buffer but only 15 μl were recovered. The cycle number for PCR amplification was then determined by RT-qPCR using 5 μl NEBNext Ultra II Q5 master mix with 0.5 μl 20X SYBR/ROX, 0.5 μl 1:10 diluted barcoded primers (NEB, E7600), 1 μl of IP or input adaptor-ligated DNA, and 3 μl nuclease-free water. The libraries were then amplified with 2 μl of barcoded primers diluted 1:10 (NEB, E7600), 20 μl NEBNext Ultra II Q5 PCR mix, 4.5 μl of IP or input adaptor-ligated DNA, and 13.5 μl nuclease-free water using the cycle number identified for each sample from the CT values found by qPCR. A 1.6x SPRI cleanup was performed followed by a tube switch. An additional 1.6x SPRI cleanup was then performed before beads were air dried and DNA was eluted in 11 μl of elution buffer. 10 μl of each sample were recovered and the KAPA Library Quantification kit was used to determine final library concentrations. 1 μl of final libraries were run on a 5% PAGE gel in 10 μl 1X loading dye to assess library fragment size distributions. Libraries were pooled at 10 nM final concentration and sequenced on an Illumina sequencer.