GSM8658392: Myotube Rep1; Homo sapiens; Hi-C
To wash the nuclei, samples were thawed and resuspended in 50 µL ice cold PB, followed by the addition of 150 µL ice-cold RNase-free water. 50 µL of Buffer C1 (Qiagen Epitect Hi-C Kit, #59971) was added to each sample and mixed. Samples were then centrifuged at 2500 x g and 4 ˚ C for 5 minutes. The supernatant was aspirated and the nuclear pellet was resuspended in 500 µL of RNase-free water, before being centrifuged again at 2500 x g and 4 ˚ C for 5 minutes. Library generation and subsequent steps used proprietary reagents from Qiagen's Epitect Hi-C Kit. Washed nuclei were digested with according to the Epitect Hi-C protocol using a proprietary enzyme cocktail that cut at the GATC motif. Nuclei were end-labeled with biotin followed by ligation for 2 hours at 16 ˚C. Ligated chromatin was then de-crosslinked using 20 µL of Proteinase K solution at 56 ˚C for 30 minutes and then heated to 80 ˚C for 90 minutes. Ligated, decrosslinked DNA was purified using a Qiagen column kit (Qiagen Epitect Hi-C Kit, #59971).  and resuspended in 130 µL EB. Hi-C library samples were fragmented to a median size of between 400 and 600 bp using a Covaris E220 sonicator with a sample size of 130 µL. Samples were purified for fragments between 400 and 500 bp using a Qiagen bead purification size exclusion kit . Reagents used in Hi-C library generation were supplied with Qiagen Epitect Hi-C Kit unless otherwise noted.  Hi-C samples were streptavidin-purified to enrich for properly ligated contact pairs using streptavidin beads and a magnetic bead rack. Beads were first washed in 100 µL of bead wash buffer, resuspended in 50 µL of bead resuspension buffer, and then mixed with 50 µL of Hi-C sample. The mixture was then incubated at room temperature for 15 minutes in a thermal mixer at 1000 RPM. Enriched bead-bound DNA was then end-repaired, phosphorylated, and poly-A tailed using a combined ER/A-tailing solution. The samples were incubated for 15 minutes at 20 ˚C followed by incubation at 65 ˚C for 15 minutes. Beads were then washed once with 100 µL of bead wash buffer, washed again with 95 µL of adapter ligation buffer, and resuspended in adapter ligation buffer in preparation for ligation of Illumina adapter sequences. Each sample was mixed with 5 µL of one of 6 Illumina adapter sequences (specified in the Qiagen protocol appendix) and 2 µL of ultralow input ligase. These samples were then incubated for 45 minutes. Following adapter ligation, each sample was washed three times before adding 400 µL of library amplification mixture to the beads. Samples were then distributed equally across 8 wells of a 96-well PCR plate and cycled using the following parameters on an Eppendorf Mastercycler x50s thermocycler