title: GSM2054408: Cwalk library from 1pg of 3C template amplified with Phi29 (hs_mesc_I); Mus musculus; OTHER
library_construction_protocol: Between 25 to 50 x 10e7 cells were used to perform in-nucleus ligation 3C; biotin fill-in was omitted. Briefly, adherent cells (mES) were incubated in the presence of trypsin (Biological Industries 03-053-1) for 8-10 minutes max at 37°C and passed through a cell strainer after harvesting to ensure a single-cell suspension. Cells (including K562 from now on) were washed 1x with phosphate-buffered saline (PBS) and resuspended PBS/FBS 10% and cross-linked in formaldehyde final concentration of 2% for 5 minutes at room temperature. After quenching with 0.125 M glycine and wash with PBS, cells were incubated in permeabilization buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2 % NP-40 alternative (CALBIOCHEM 492016), Protease Inhibitor Cocktail (P8340 Sigma)) for 60 minutes at 4°C while slowly mixing. Cells were split into 5 M cell aliquots and washed 2x with cold PBS. After the second wash, cell pellets were then flash frozen on liquid nitrogen. Pellets were resuspended on 175.5 µl H2O and 24.5 µl DpnII RE buffer 10x. After addition of 3 µl of 20% SDS (final concentration of 0.3%), cells were incubated for 60 minutes at 37°C while agitating (750 RPM). Triton X-100 20% was added to a final concentration of 1.8% and incubated for 60 min at 37°C. Aliquots were then digested with 15 µl HC DpnII (NEB 50,000 U/ml) ON. Cells were centrifuged for 5 minutes at 600 g and washed 1x with PBS. Incubation of pellets for 20 minutes at 65°C was performed in order to heat-inactivate any DpnII residue. In-nucleus ligation was then performed incubating cells on ligation mix (415 µl H2O, 5 µl BSA (10mg/ml NEB) and 5 µl T4 ligation enzyme NEB (2x106 U/ml)) ON at 16°C.  Cross-link was reversed by incubating aliquots with 10 mg/ml proteinase K (Roche) at 65°C ON followed by the addition of 15 µl of RNase A (Roche) for 45 minutes at 37°C. DNA was purified with AmpureXP SPRI beads 1x (Beckman Coulter; A63881) with two elution rounds of 100 µl each with the aim of size selecting for the high molecular weight portion of the sample. C-walks. Serial dilutions (1:10) of 3C template were performed to reach a final concentration of 10 pg/µl stock template. REPLI-g single cell kit (Qiagen 150343) protocol for purified genomic DNA was used under the recommendation of the manufacturers with a few modifications. To avoid contamination, all operations before the amplification step were performed under strict hygienic conditions (TC hood), thorough DNA decontamination with DNAZap (ThermoFisher) and UV irradiation of laboratory utensils. A single C-walk amplification reaction consisted of ~1 picogram of 3C material. Typically for 100 reactions, 10 µl of 10 pg/µl stock were mixed into 16.56 µl of sterile TE buffer and denatured at RT for 3 minutes by mixing with buffer D1 (5.83 µl DLB buffer + 20.83 scH2O; both from REPLI-g sc kit). Denaturation of DNA was stopped by addition of 50 µl buffer N1 (7.5 µl Stop solution + 42.5 µl scH2O; both REPLI-g sc kit) and stored on ice. A final Master Mix was prepared by mixing 400 µl of reaction buffer (290 µl Reaction Buffer + 90 µl scH2O + 20 µl Phi29 DNA polymerase; from REPLI-g sc kit). Alternatively, the enzyme can be substituted with NEB Phi29 (M0269S) using the same volume. An aliquot of 5 µl of the Master Mix was taken as negative control. Master Mix and DNA template were then mixed and kept on ice. Aliquots of 5 µl from the Master Mix were split into single wells on 96-well plates and incubated for 2 hours and 50 minutes at 30°C followed by a heat inactivation step of 3 minutes at 65°C. Additional 45 µl of H2O were added to each well after spinning down for a few seconds. The amplified material was cleaned with SPRI beads (2.5x) and resuspended in 100 µl. Phi29-amplified material (300-700 ng per library) was sonicated in Bioruptor (program: 11 cycles of 5s on, 90s off) in 0.1 ml Bioruptor Microtubes (C30010015) to reach a DNA fragment size between 900-1,500 bp, peaking at 1,200 bp. The sonicated DNA (42.5 µl) was then subjected to end-repair reaction (5µl 10x end-repair buffer + 2.5 µl end-repair mix (NEB E6050L)) at 20°C for 30 minutes. The DNA was cleaned with SPRI beads (2.2x) and eluted in 38µl EB. A-tailing reaction was performed with 5 µl NEB buffer 2, 4 µl Klenow (NEB M0212M) and 10 µl dATP 10nM for 30 minutes at 37°C, cleaned with AmpureXP beads (2.2x) and eluted in 30 µl EB. DNA template was ligated with 5 µl Illumina indexed adapters 0.75 µM, 5 µl T4 quick ligase (NEB M2200) and 40 µl 10x ligase buffer for 15 minutes at 25°C. Ligated template was then cleaned with SPRI beads (1.3x) and eluted in 41 µl. Each indexed template was PCR amplified on a final volume of 50 µl with the following reagents: 1 µl dNTPs (10 mM), 2 µl Illumina enrichment primer I and II mix 10mM, 1 µl Pfu ultra II Fusion HS DNA polymerase (Promega 600670), 5 µl Pfu ultra II Fusion buffer (Promega 600670) and 200 ng of Phi29-amplified material. PCR program: 2 minutes 95°C, 14 cycles of 30s 95°C, 30s 55°C, 60s 72°C, final extension of 10 minutes 72°C. Following the PCR reaction, products were cleaned with SPRI beads (1x) and quantified. Libraries were pooled in groups of 8 in equal quantities and ran on a 1% agarose gel, not loading more than 1000 ng per lane. Gel was excised selecting for an 800-1200 bp band. DNA was purified using minelute gel extraction kit (QIAGEN). Samples were then pooled all together and paired-end sequenced using the Illumina platform. 3way 4C.  5-10 µg of 3C template was taken to a volume of 200 µl Elution Buffer (EB, 10mM Tris-HCl, pH 8) and was sonicated at 4°C using Bioruptor; program: 5 cycles of 25s on, 60s off. The sonicated material was inspected by running 10 µl on agarose gel to assure size distribution of 700-900 bp. The sonicated DNA was subjected to end-repair reaction: 20µl 10x end-repair buffer and 10µl end-repair mix (NEB E6050L) were added to the mix and incubated at 20°C for 30 minutes. The DNA was cleaned by SPRI (2.2x) beads and eluted in 76 µl EB. A-tailing reaction was performed with 10 µl NEB buffer 2, 4 µl Klenow (NEB M0212M), and 10 µl dATP 10 nM. Following A-tailing, the 5’ ends of the DNA were dephosphorylated with 2 µl of Alkaline Phosphatase (NEB M0290S) at 50°C for 60 minutes, and the DNA was cleaned with SPRI beads (2x), then eluted in 67 µl EB. The DNA template was ligated in 1,000 ng aliquots with 5 µl Illumina indexed adapters 15 µM (final concentration 0.4 µM), 10 µl quick ligase mix and 80 µl 10x buffer (NEB M2200). To release the non-ligated strand of the adapter, the DNA was denatured at 95°C for 3 minutes, moved immediately to ice and cleaned with SPRI beads (1x) to select for the UMI-4C DNA template . Two nested PCR reactions were used for UMI-4C library construction. The PCR reactions were performed in 50 µl volume with the following reagents: 10 µl GoTaq Flexi buffer (Promega M792A), 3 µl MgCl 25mM (Promega A3511), 25 µM dNTPs, 2 µl bait primer pool 100 µM (final concentration of each bait varies depending on the pool size), 2 µl Illumina enrichment primer II 10 mM (0.4 mM final concentration), 1 µl GoTaq (Promega M3008) and 200 ng UMI-4C DNA template. PCR program: 2 minutes 95°C, 20 cycles of 30s 95°C, 30s 56°C, 60s 72°C, final extension of 5 minutes 72°C. Following the PCR reaction, products were cleaned with SPRI beads (1x). The second PCR reaction was identical to the first with the only difference on the primer pool. Second primer targets a nested sequence of the 4C bait and is attached with the Illumina I adapter. The program for the second PCR reaction was the same but performed for only 18 cycles.The amplified DNA was cleaned with SPRI beads (0.7x) and the size distribution was inspected using Tapestation D1000 (Agilent Technologies). Typically, libraries had a size distribution around 800-1000 bps. The libraries were then pooled to 10 nM and were multiplexed with other libraries for paired-end sequencin
attributes.tag: GEO Accession
attributes.value: GSM2054408