GSM6597857: MacroH2A1.1 (1C); Homo sapiens; OTHER
Cells on the fourth day post-transfections were collected in 1.5mL tubes. Cut & Tag was performed according to the manufacturer's protocol (Active Motif, California, USA). Briefly, cells were harvested and washed once using 1mL of 1x Wash buffer. In parallel, the Concanavalin A Beads were prepared. Each 500.000 cells, 20µL of the beads' slurry were withdrawn and mixed with 1,6mL of 1x Binding buffer. Beads were harvested using a magnetic stand and washed twice with 1,5 mL of 1x Binding buffer. The pellet containing the cells previously harvested and the beads' suspension were mixed in a 1.5 mL tube, rotating end-over-end for 10 minutes at room temperature. Cells were harvested by using a magnetic stand to clear and the supernatant was discarded. The pellet was resuspended in 50µL of Antibody Buffer and 1µL of Rabbit ab9108 Anti-6X His tag antibody (Abcam, Cambridge, United Kingdom) was added where needed. The mix was incubated overnight at 4oC with orbital mixing ensuring the liquid to stay together at the bottom of the tube. The Guinea Pig Anti-Rabbit Antibody secondary antibody was diluted 1:100 in Dig-Wash Buffer. The tubes were cleared using a magnetic stand and the liquid was discarded. 100µL of Guinea Pig Anti-Rabbit Antibody mix was added and tubes were incubated at room temperature for 60 minutes in an orbital rotator. Cells were cleared through a magnetic stand, and they were washed three times using 1mL of Dig-Wash Buffer. In parallel, the CUT&TAGTM Assembled pA-Tn5 Transposomes was diluted 1:100 with Dig-300 Buffer. For each immunoprecipitated samples, 100µL of CUT&TAGTM Assembled pA-Tn5 Transposomes was added, and the mix was placed on an orbital rotator for 60 minutes at room temperature. The cells were harvested using a magnetic stand and eventually washed three times using 1mL of Dig-300 buffer. In the following step each tube was supplemented with 125µL of Tagmentation Buffer and incubated at 37oC for 60 minutes. To isolate the DNA surrounding macroH2A1.1 and macroH2A1.2 deposition regions, the tagmentation was first stopped by addition of 4,2µL of 0,5M EDTA, 1,25µL of 10% SDS and 1,25µL of Proteinase K (10mg/mL) per each sample. The tubes were then vortexed at full speed and incubated for 60 minutes at 55oC to digest. The samples were cleared using a magnetic stand and 625µL of DNA Purification Buffer per each were added. The samples were transferred to DNA Purification Columns, centrifuged at 17000G for 1 minute. After the flow-through was discarded, 750µL of DNA Purification Wash Buffer was added to each sample, which was then centrifuged again at 17000G for 1 minute. The flow-through was discarded. To remove any DNA Purification Wash Buffer leftover, the empty columns were further centrifuged at 17000G for 2 minutes. To isolate the DNA, 35µL of DNA Purification Elution Buffer were added to each sample, which was then incubated at room temperature for 1 minute and eventually centrifuged at 17000G for 1 minute. Library preparation for Cut & Tag samples was performed according to the manufacturer's protocol (Active Motif, California, USA). Paired-end Illumina sequencing was obtained using Illumina Hiseq 2500 (Illumina, San Diego, CA,  USA), obtaining two FASTQ files for each of the two biological replicates of macroH2A1.1,  two for each of the three biological replicates of macroH2A1.2  and two for each of the three biological replicates of the control samples, for a total of 4.8, 8.1 and 7.2 million reads, respectively.