GSM8269304: RES.oxa.damage.3hrs.rep3; Homo sapiens; OTHER
For in-situ Hi-C, proximal ligated DNA is purified according to manufacturer's protocol in Arima-HiC kit (Arima Genomics, #A510008, Document #A160134 v01). For damage-seq, cell pellet is resuspended in 200uL PBS with 10uL RNase A (Thermo #EN0531), 20uL Proteinase K and 200uL Buffer AL (both are from QIAGEN #69504). The suspension was then incubated at 55°C for 20 mins, followed by purification with the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN #69504) according to manufacturer's instruction. In-situ Hi-C and Damage-seq For in-situ Hi-C, HCT116 OxR control cells Hi-C library was generated with the Arima-HiC kit (Arima Genomics, #A510008, Document #A160134 v01) and Arima Library Prep Module (Arima Genomics, Cat #A303011, Document #A160432 v02) following manufacturer's instruction. For oxaliplatin damage-seq, cell pellet is resuspended in 200uL PBS with 10uL RNase A (Thermo #EN0531), 20uL Proteinase K and 200uL Buffer AL (both are from QIAGEN #69504). The suspension was then incubated at 55°C for 20 mins, followed by purification with the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN #69504) according to manufacturer's instruction. The purified genomic DNA is subsequently sonicated into 200-700bp fragments with Covaris sonicator. The fragmented genomic DNA (~2ug) was used for End repair, 5' phosphorylation and dA-Tailing using 3uL End Prep Enzyme Mix and 7uL End Prep Reaction Buffer (NEB #E7546 or as included in NEB #E7645), at 20°C for 30 mins and then 65°C for 30 mins, before cooling down to room temperature. Following this, annealed AD1 adapter (500 pmol) was ligated to End Prepped DNA with 30uL Ligation Master Mix and 1uL Ligation Enhancer (NEB #E7595 or as included in NEB #E7645) at 16°C overnight. AD1 ligated DNA was purified with 1.2x AMPure XP beads and eluted in 50uL nuclease free water. Before the incubation with antibody coated beads, AD1 ligated DNA was denatured into ssDNA with 20uL 8M urea at 95°C for 1min, then placed on ice for 1min, and immediately incubated with antibody coated beads. To prepare the antibody coated beads, 40uL anti-rat Dynabeads (Thermo, #11035) was washed three times with 1mL 1x PEXB buffer (1x PBS, 2mM EDTA, 0.01% Triton X-100 and 0.025% albumin). The beads were then incubated with 10ug salmon sperm DNA (Thermo, #AM9680) and 1.5uL anti-cisplatin modified DNA antibody (Abcam, #ab103261) in 100uL 1x PEXB buffer at 4°C with rotation for 3 hours. After incubation, antibody coated beads were then washed three times by 1mL 1x PEXB buffer and incubated with denatured, AD1 ligated DNA and 10ug salmon sperm DNA overnight at 4°C, to capture the oxaliplatin damaged DNA. The beads were then washed subsequently with 400uL Wash Buffer U (20mM Tris-HCl (pH=8), 2mM EDTA, 1% Triton X-100, 2M urea), 400uL Wash Buffer II (20mM Tris-HCl (pH=8), 2mM EDTA, 1% Triton X-100, 0.5M NaCl, 0.1% SDS), 400uL Wash Buffer III (10mM Tris HCl (pH=8), 1mM EDTA, 150mM LiCl, 1% IGEPAL CA-630, 1% Na deoxycholate), 400uL Wash Buffer IV (100mM Tris-HCl (pH=8), 1mM EDTA, 500mM LiCl, 1% IGEPAL CA-630, 1% Na deoxycholate), and 400uL 1x TE (10mM Tris-HCl, 1mM EDTA). The oxaliplatin damaged DNA fragments were then eluted from beads with 100uL Elution buffer (50mM NaHCO3, freshly prepared, 1% (w/w) SDS) at 65°C for 10 mins. The eluted DNA was then precipitated by adding 10uL 3M sodium acetate solution, 1uL glycogen (Thermo #R0561), and 300uL ethanol at -80°C overnight. Ethanol precipitated DNA is dissolved in 7uL nuclease free water. To this solution, Bio3U primer (30 pmol), 10uL Q5 Master Mix (NEB #M0544 or included in NEB #E7645) and nuclease free water were added to reach a total volume of 20uL. Primer extension is performed at 98°C for 45s, followed by 65°C for 5 min, and then placed on ice for cooling. Next, 2uL Exo I (NEB, M0293S) was added to remove the excessive primers at 37°C for 10 min. The primer extended DNA is then purified with 2x AMPure XP beads, and eluted with 30uL water. The DNA elute was denatured at 95°C for 90s, and then immediately placed on ice for 2 min. The denatured DNA is subsequently incubated with 5uL Streptavidin C1 beads (Thermo, #65001) in 30uL 2x B&W buffer (B&W final concentration (1x): 5mM Tris HCl (pH=7.5), 1mM EDTA, 1M NaCl) at 4°C for 1 hour to allow the biotinylated dsDNA being captured by C1 bead. The bead is then washed first with 100uL 1x B&W buffer, and then with 100uL 1x TE buffer. Finally the DNA was released from C1 beads into 18uL 0.1x TE by adding 2uL USER enzyme (NEB, #M5505) and incubating at 37°C for 25 min. The beads were washed by 25uL 1x B&W, and the supernatant was combined with the 20uL DNA elute obtained from the previous step. To eliminate Bio3U extended DNA strand that read through the intact DNA fragment without damage (subtractive hybridization), Oligo SH (20 pmol) was added, followed by denaturing at 95°C for 1 min and naturally cooling down to room temperature to allow hybridization.  Oligo SH hybridized product was then captured by adding 10uL Streptavidin C1 beads (resuspended in 60uL 1x B&W) for 1 hour at 4°C. DNA was purified by ethanol precipitation at -80°C overnight. To added second adapter to the 3'end, purified DNA was incubated with annealed AD2 (40 pmol) in 10uL 1x hybridization buffer (10mM Tris‐HCl, pH7.5,100mM NaCl, 0.1mM EDTA) at 65°C for 10min, and then 16°C for 5min. After adding 10uL ligation mix (From Thermo #EL0013, 2uL 10x ligation buffer, 1uL T4 DNA ligase (HC), 1uL 50% PEG solution, 1uL nuclease free water), the AD2 ligation reaction is incubated at 16°C overnight. The ligation product was then purified by 2x AMPure XP beads and PCR amplified by Q5 Master Mix (NEB #M0544 or included in NEB #E7645S) for 13-16 cycles. The PCR products were purified with AMPure XP beads.