GSM8999223: DNMT1_KO_rep_2_early_repli_seq; Homo sapiens; OTHER
Repli-seq data was generated from cells at approximately 70% confluency to ensure most of the cells were actively cycling. To label DNA, cells were given fresh media supplemented with 100μM 5-Ethynyl-2'-deoxyuridine (EdU) and incubated at 37°C for 30 minutes. Cells were then collected by trypsinization along with media and a PBS wash of the flask to ensure non-adherent mitotic cells were not lost. The cell suspension was then centrifuged at 200g for 5 minutes and washed with 10ml PBS before being resuspended in 2.5ml PBS supplemented with 1% FCS. Cells were fixed by gently adding 7.5ml of 100% ethanol to a final concentration of 70% with gentle vortexing followed by incubation on ice for 30 mins and storage at –20°C. Fixed cells were washed with 5ml ice cold PBS, and then resuspended in 5ml 100mM HCl and 0.015% pepsin (0.75mgr) in H2O before being incubated at 37°C for 30 minutes while rotating. Following centrifugation at 600g for 10 minutes, pellets were washed with 5 ml ice cold PBS and then resuspended in 1ml PBS along with 50μl Propidium Iodide (PI) and 20μl RNase A and incubated at room temperature for 30 minutes while protected from light. Extracted Nuclei were counted using a hematocytometer and suspended to approximately 3x106 nuclei/ml to ensure optimal flow during FACS sorting. Nuclei were sorted on a FACSAria flow cytometer (BD Biosciences) in PBS supplemented with 0.25% BSA based on their DNA content using PI staining, into three fractions: Early, Middle and Late S-Phase (defined by equally splitting the window between the G1 and G2 peaks into three equal portions). DNA was then extracted from sorted nuclei as described above except they were sonicated on a Bioruptor following proteinase K digestion (15 cycles of 30 seconds on – 30 seconds off, on high at 4°C). Precipitation of extracted DNA was then performed with linear acrylamide (LPA) carrier and isopropanol and resuspended in TE. DNA was then quantified using Qubit High Sensitivity reagents and sonicated on a Covaris E220 sonicator to yield 100-300bp fragments which were quality controlled on a Bioanalyser. Re-precipitation of DNA was was then performed with LPA carrier and ethanol before being resuspended in water. A click reaction was then performed to conjugate biotin to the EdU by adding 3ul 2M TAB (0.4M), 30ul DMSO, 6ul 5mM Ascorbic Acid (2mM), 1.2ul 10mM Biotin Azide (0.8mM), 3ul 10mM CuTBTA (2mM) and 1.8ul H2O. Samples were then incubated overnight at room temperature while covered from light and cleaned up by ethanol precipitation with LPA carrier. EdU containing DNA was then enriched using Myone C1 Streptavidin Dynabeads (Invitrogen) and a magnetic rack overnight at 4°C with rotation. Following this, the samples were washed at 4°C with: TSE-I (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton, 0.1% (v/v) SDS) x2, TSE-II (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton, 0.1%(v/v) SDS) x1 and TE x1. Samples were eluted by adding water at 95°C and incubation with vortexing at 95°C for 10 mins. Successful enrichment of early and late replicating DNA was then validated by qPCR of early (BMP1, TEK, MELK, RTTN) and late replicating (NETO1, CDH8, DPPA2 and PTPRD) loci, based on a published protocol and HCT116 WT repli-seq data (Du et al., 2021; Marchal et al, 2018). Primer sequences used in this validation are included in Supplementary Table 2. Repli-seq libraries were then prepapred using the Acel-NGS 1a Plus DNA library kit along with the 1A Plus Set A Indexing Kit following the manufacturer's instruction (Swift Biosciences). However, as our DNA template contain Uracil due to the EdU incorporation, the kit's polymerase, and subsequent buffer (Reagent W2, Buffer W3 and Enzyme W4) were replaced with KAPA HiFi HotStart Uracil+ Ready Mix Kit (Roche), which included an uracil tolerant polymerase. Libraries were assessed for size distribution on the Agilent Bioanalyser (Agilent Technologies) and quantified using the Qubit 2.0 Fluorometer. Repli-seq libraries were sent for whole genome sequencing at the Wellcome Trust Clinical Research Facility (WTCRF). The libraries were sequenced using the Illumina NextSeq 500/550 High-Output v2.5 (75 cycles) Kit on the Illumina NextSeq 550 platform over 2 flow cells to give 75bp single-end reads.