GSM5768741: H1_P5k27_sc2for1_rep3_(20211206_DJ_Hs_H1_2for1); Homo sapiens; OTHER
Single-cell CUT&Tag2for1 was performed using lightly fixed K562 and H1 nuclei. Frozen nuclei were thawed and aliquots containing 20,000 nuclei were centrifuged at 700 x g for 4 minutes at 4˚C. Nuclei were washed once with Wash buffer, centrifuged again, and then resuspended in Antibody buffer (10 mM HEPES pH 150 mM NaCl, 2 mM spermidine, 2 mM EDTA, 0.1% BSA, and Roche complete EDTA-free protease inhibitor) with primary anti-Pol2S5p antibody (Cell Signaling Technology cat. no. 13523, 1:50) and anti-H3K27me3 (Cell Signaling Technology cat. no. 9733, 1:100) in 0.6 mL PCR tubes. Primary antibody binding was performed overnight at 4oC. Samples were centrifuged at 700 x g for 4 minutes at 4˚C between incubation steps. and incubated for 1 hour at room temperature for the guinea pig anti-rabbit secondary antibody (Antibodies Online cat. no. ABIN101961 1:100) and for 1 hour at room temperature for pAG-Tn5 (Epicypher cat. no. 15-1117, 1:20) tethering. Samples were then centrifuged, washed with 300-wash buffer, pelleted by centrifugation, and then resuspended in 5 mM MgCl2, 10% hexanediol, 10 mM TAPS pH 8.5 for 20 min at 37˚C for tagmentaion. Reactions were stopped by adding EDTA to a final concentration of 1 mM, and kept at 4 ˚C until dispensation on the ICELL8 platform. Cells were processed on the ICELL8 instrument according to a previously optimized protocol for release of tagmented DNA in SDS, followed by a Triton X-100 neutralization step and PCR amplification (Janssens et al., 2021). Briefly, the volume of 10 mM TAPS Buffer pH 8.5 was adjusted to 65 uL per 20,000 nuclei to yield a concentration of ~300 nuclei/uL and nuclei were stained with 1X DAPI and 1X secondary diluent reagent (Takara Cat# 640196). The 8 source wells of the ICELL8 were loaded with 65 uL of the suspension of tagmented nuclei and dispensed into a SMARTer ICELL8 350v chip (Takara Bio, cat. no. 640019) at 35 nL per well. The chip was then sealed for imaging and spun down at 1200 x g for 1 min. Imaging on a DAPI channel confirmed the presence of single cells in specific wells. Non-single-cell wells were excluded from downstream reagent dispenses. A volume of 35 nL of 0.19% SDS in 10 mM TAPS Buffer pH 8.5 was dispensed into active wells and the chip was dried, sealed and spun down at 1200 x g for 1 min. The chip was placed in a thermocycler and held at 58oC for 1 hr to release tagmented chromatin. The chip was spun at 1200 x g for 1 min before opening, and 35 nL of 2.5% Triton X-100 in ddH20 was dispensed into all active wells. To index the whole chip, 72 × 72 i5/i7 primers containing unique indices (5,184 microwells total) were dispensed at 35 nL in wells that contained single cells, followed by two dispenses of 50 nL (100 nL total) KAPA PCR mix (2.775 X HiFi Buffer, 0.85 mM dNTPs, 0.05 U KAPA HiFi polymerase / µL, Roche Cat# 07958846001). The chip was sealed for heated incubation and spun down at 1200 x g for 1min after each dispense. PCR on the chip was performed with the following protocol: 5 min at 58 °C, 10 min at 72 °C and 2 min at 98°C, followed by 15 cycles of 15s at 98°C, 15s at 60°C and 10s at 72°C, with a final extension at 72 °C for 2 min. The contents of the chip were then spun out into a collection tube (Takara Cat# 640048) at 1200g for 3 min. Two rounds of SPRI bead cleanup at a 1.3 : 1 v/v ratio of beads to sample were performed to remove residual PCR primers and detergent. Samples were resuspended in 20 uL of 10 mM Tris-HCl pH 8.0. Library quality and concentration were evaluated by Agilent Tapestation capillary gel analysis, and single-cell CUT&Tag2for1 samples were then pooled with bulk libraries prepared using compatible barcodes. PE25 sequencing was performed on an Illumina HiSeq2500 by the Fred Hutch Genomics Shared Resource. CUT&Tag2for1 was carried out using pA-Tn5-adapter-antibody conjugate complexes to integrate barcoded adapters via tagmentation directly to DNA for each antibody target on both sides of the insert, so that the DNA that is released is already a barcoded library.