GSM8208135: MTA2_CR_HDP2_Day7_delZnF1; Homo sapiens; OTHER
ATAC-seq: ATAC-seq was performed with the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit, as previously described (Buenrostro et al., 2015). 50,000 cells were collected and washed once with cold PBS. Cells were then permeabilized with 50 μL ice-cold lysis buffer containing10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-360 for 10 minutes. The transposition reaction was carried out at 37°C for 90 minutes in 50 μL volume containing 25 μL TD Buffer and 2.5 μL TDE1 Tagment DNA Enzyme. Immediately following transposition incubation, DNA was purified with the MinElute PCR Purification Kit (Qiagen).  CUT&RUN was performed as previously described (Liu et al., 2021). Briefly, 1x106 cells were freshly collected for each sample. Cells were washed once with cold PBS and processed according to the following steps: (1) 800 μL of NE buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol and protease inhibitor (Roche)) was added to the pellet for nuclear isolation. Nuclei were pelleted at 600g for 3 min and resuspended in 200 μL of NE buffer. (2) 30 μL of BioMagPlus Concanavalin A bead slurry was washed twice and resuspended in 100 μL of binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2). Beads were then added to the nuclear suspension. The mixture was thoroughly mixed on a rocker for 10 min at RT to allow binding of nuclei to the beads. The nuclei were pelleted with a magnet stand and blocked with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA and protease inhibitor (Roche)) supplemented with 2 mM EDTA for 5 min on ice. (4) After washing once with wash buffer, nuclei were resuspended in 200 μL of wash buffer containing 1 μg of antibody and incubated overnight at 4°C. (5) The next day, nuclei were washed twice on a magnet stand with wash buffer and incubated with 1:1000 Protein A-micrococcal nuclease fusion protein (pA-MNase) in 200 μL of wash buffer for 1 hr at 4°C. The nuclei were then washed twice with wash buffer to remove unbound pA-MNase. (6) Nuclei were resuspended in 150 μL of wash buffer and chilled on a metal block at 0°C in a water-ice mixture. 3 μL of 100 mM CaCl2 was added to the nuclei to activate pA-MNase with incubation at 0°C for 60 min. (7) The reaction was stopped by the addition of 150 μL of 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complex was released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometry and bioanalyzer quality control. The antibodies used for CUT&RUN were BCL11A (Sigma, ZRB1518), NF-YA (Abcam, ab139402) and MTA2 (Abcam, ab8106). RNA: Total RNA was isolated from cells using the RNeasy Plus Mini Kit (Qiagen). ATACseq: Transposed DNA fragments were amplified using NEBNext® Ultra™ II Q5 Master Mix and PCR reaction was carried out as follows: 72°C for 5 min; 98°C for 30 s; 8 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. The resulting libraries were purified using the QIAquick PCR Purification Kit (Qiagen), quantified with Qubit fluorometer and TapeStation, and then sequenced using the NextSeq 500 platform. Paired-end sequencing was performed with read length of 42 bp and index of 8 bp. CUT&RUN: Library preparation of CUT&RUN was performed using a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, as previously described (Liu et al., 2021). Briefly, 6 ng of DNA was used as input. End preparation was performed at 20 °C for 30 min and then 50 °C for 60 min. 0.6 pmol adapter was added and ligated to end-preparation products at 20 °C for 15 min. USER enzyme was then added to cleave the uracil in the loop at 37°C for 15 min. 1.7x volume of AMPure XP beads was used to purify the ligation product for efficient recovery of short fragments. To amplify the library, the ligation product was mixed with 2x NEBNext® Ultra™ II Q5 Master Mix, universal primer and index primers. PCR reaction was carried out as follows: 98°C for 30 s; 12 cycles of 98°C for 10 s and 65°C for 10 s; and a final extension at 65°C for 5 min. The PCR product was subjected to double-size selection, first with 0.8x and then with 1.2x AMPure XP beads. The purified library was quantified by Qubit fluorometer and TapeStation. Libraries were pooled in equimolar ratios and sequenced using the NextSeq500 platform. Paired-end sequencing was performed with read length of 42 bp and index of 6 bp. RNAseq: RNA sequencing libraries were prepared using the KAPA mRNA HyperPrep Kit (Roche) from 200 ng of purified total RNA. The finished dsDNA libraries were quantified by Qubit fluorometer, TapeStation, and RT-qPCR using the KAPA Library Quantification Kit (Roche). Uniquely indexed libraries were pooled in equimolar ratios and sequenced on Illumina NextSeq500 with single-end 75bp reads. ATACseq and CUT&RUN: read length 42 RNAseq: read length 75