title: GSM6203832: Promoter Capture Micro-C, C42B, rep2; Homo sapiens; OTHER
library_construction_protocol: The human prostate cancer C42B cells were obtained from ViroMed Laboratories (Minneapolis, MN, USA) Cells were grown at 37 °C in 5% CO2. It was grown in RPMI1640 culture medium, and supplemented with 10% fetal bovine serum (Gibco by Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin. All cell stocks were authenticated at the USC Norris Cancer Center cell culture facility by comparison to the ATCC and/or published genomic criteria for that specific cell line; all cells were documented as free of mycoplasma. 1 x 10^6 cells were harvested, and were cross-linked with formaldehyde for 10 minutes at room temperature. Cells were digested with MNase Enzyme Mix and were lysed with 20% SDS. Lysate was mixed with Chromatin Capture Beads to bind chromatin and incubated at room temperature for 10 minutes. End Polishing Master Mix was added lysate and incubated for 30 minutes at 22*C and 30 minutes at 65*C to end polish. For ligation, 50ul of Bridge Ligation Mix and Bridge Ligase were added and incubated at 22ºC for 30 minutes, and 50ul of Intra-aggregate Ligation Buffer and Intra-aggregate Ligation Enzyme Mix were added and incubated at 22ºC for 1 hours. DNA were isolated by reverse-crosslinking with Proteinase K and Crosslink Reversal Buffer, and incubating at 55ºC for 15 minutes, 68ºC for 45 minutes. DNA was then purified. For library preparation, purified DNA was added with End Repair Master Mix and incubated at 20ºC for 30 minutes and 65ºC for 30 minutes for end repair. adaptor for Illumina, ligation enzyme mix and ligation enhancer were added for adapter ligation, then DNA was purified. Adaptor-ligated DNA was then washed and captured with Streptadvidin beads. PCR was performed, and size range between 350-1,000bp was selected for the library. Prepared libraries were then sequenced with Illumina sequencers in pair-end mode. Micro-C libraries were pooled for hybridization reactions. For hybridization reaction, the probe solution was first heated to 95°C for 2 minutes in a thermal cycler with a lid at 105°C. Library was heated in similar way for 5 minutes. After cooling in ice for 5 minutes, and equilibrating to room temperature for 5 minutes, the probe solution was added to the library. Hybridization Enhancer was then added and the mixed solution was incubated at 70° for 16 hours in a thermal cycler with the lid at 85°C. After hybridization steps, the hybridized targets were washed and captured with Streptavidin beads, amplified with PCR, and then purified. It was sequenced on Illumina platform in pair-end mode.