organism: Mus musculus
description: Protocols: 3. We performed in situ Hi-C as described before (Díaz et al., 2018) for E14-Tir1, Ring1B-AID and Scc1-AID samples with 100K cell as input material. Mainly, differences were related to adjustments on the volume of the reactions, a shortening of the digestion step, removal of biotin from the unligated fragments, and an alternative strategy for size-selection during library preparation. 1. Wild type E14 cells were grown on gelatin-coated plates in DMEM (Gibco, Carlsbad, CA) supplemented with 10% FBS, 10 ng/mL leukemia-inhibitory factor, penicillin/streptomycin, beta-mercaptoethanol, L-glutamine and non-essential amino acids. 2. pSpCas9(BB)-2A-Puro (PX459) was a gift from Feng Zhang (Addgene plasmid # 48139). Rosa-Tir1 mES cells were generated by cotransfection of pX459 Rosa26 and pUC19 Rosa-Tir1. Transfected cells were then selected with puromycin (1 µg/ml) for two days. When colonies were large enough they were picked and screened by PCR. AID-Ring1B and Scc1-mAID-EGFP was then introduced into this cell line by the same procedure. In order to induce the degron, a 6-hour auxin treatment was conducted on the “treated” samples. 5. Phenol-Chloroform-Isoamyl alcohol (25:24:1; Sigma-Aldrich) extracted DNA was and resuspended in 30 μL of Tris pH 8.0 (Applichem) and incubated for 15 min at 37°C with RNAse A (1 μL; Applichem) to eliminate traces of RNA. In order to remove biotin from unligated fragments, DNA samples were incubated at 20°C for 4h without rotation in a mix of 10 μL of 10x NEB2 buffer (New England Biolabs), 1 mM of a dNTPs mix (10 μL), 20 mg/mL BSA (0.5 μL), 3 U/μL T4 DNA polymerase (5 μL; New England Biolabs) and nuclease-free water (up to 100 μL). Samples were sheared using a Covaris S220 instrument (2 cycles, each 50 sec long, 10% duty, 4 intensity, 200 cycles/burst). Biotinylated fragments were pulled down using Dynabeads MyOne Streptavidin C1 beads. Bead-bound libraries were end-repaired using the NEBNext Ultra End Repair module (New England Biolabs). Adaptor for Illumina sequencing was added using the NEBNext® Ultra™ dA-Tailing module (New England Biolabs). Beads were resuspended in 50 μL 10 mM Tris pH8.0. Final amplification of the libraries was done in 4 parallel reactions per sample as follows: 10 μL of the bead-bound libraries, 25 μL of 2x NEBNext Ultra II Q5 Master Mix, 5 μL of 10 μM Universal PCR primer, 5 μL of 10 μM Indexed PCR primer and 10 μL of nuclease-free water. Samples were individually barcoded as follows: Tir1+Aux_Batch1 #2, Ring1B+Aux_Batch1 #4, Scc1+Aux_Batch1 #12, Tir1+Aux_Batch3 #19, Ring1B+Aux_Batch3 #21, Scc1+Aux_Batch3 #23; and amplified for 10 (Tir1+Aux_Batch1, Ring1B+Aux_Batch1, Scc1+Aux_Batch1), 12 (Ring1B+Aux_Batch3) or 14 (Tir1+Aux_Batch3, Scc1+Aux_Batch3) cycles following the program: 98°C for 1 min, (98°C for 10 s, 65°C for 75 s, ramping 1.50°C/s) repeated 10-14 times, 65°C for 5 min, 4°C hold. Following the amplification, the four reactions were combined into one tube and size-selected using Ampure XP beads (Beckman Coulter). Final Hi-C libraries were quantified using Qubit dsDNA HS assay kit on a Qubit (Thermo Fisher Scientific) and a DNA HS kit on a 2100 Bioanalyzer instrument (Agilent). 4. Briefly, after isolation, cells were crosslinked on 37% Formaldehyde (1% final concentration; v/v) and incubated 10 min at room temperature with gentle rotation (20 rpm). The reaction was quenched by adding 2.5M Glycine solution (0.2M final concentration) and incubating 5 min at room temperature with gentle rotation (20 rpm). Cells were pelleted and resuspended on 1mL cold 1x PBS twice. After the last spin, cells were gently resuspended in 250 μL of ice-cold in situ Hi-C buffer (10 mM Tris-Cl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, cOmplete Ultra protease inhibitors) and incubated on ice for 15 min. Samples were then spun (300 g for 5 min at 4°C) and resuspended in 250 μL of in situ lysis buffer. Cells were pelleted (13,000 g for 5 min at 4°C), gently resuspended in 250 μL ice-cold 10x NEB2 buffer. Permeabilisation of nuclei was done by resuspending them in 50 μL of 0.4% SDS and incubating at 65°C for 10 min. Quenching of the SDS was done by adding 25 μL of 10% Triton X-100 and 145 μL of nuclease-free water, respectively, and incubated at 37°C for 45 min with rotation (650 rpm). Then chromatin digestion was done by adding 100 U of MboI in 20 μL of 10x NEB2.1 buffer (New England Biolabs) for 90 min at 37°C with gentle rotation. MboI was heat-inactivated by incubating 20 min at 62°C. The overhangs generated by the restriction enzyme digestion were filled-in by adding a mix of 0.4 mM biotin-14-dATP (18.75 μL; Life Technologies), 10 mM dCTP/dGTP/dTTP (0.75 μL of each dinucleotide), and 5 U/μL DNA polymerase I Klenow (8 μL; New England Biolabs), and incubating for 90 min at 37°C with gentle rotation. DNA fragments were ligated using a mix of nuclease-free water (657 μL), 10x T4 DNA ligase buffer (120 μL), 10% Triton X-100 (100 μL), 20 mg/mL BSA (12 μL) and 5 Weiss U/μL T4 DNA ligase (5 μL added in two times; Thermo Fisher Scientific) by incubating 4h at 20°C with gentle rotation. Nuclei were spun down (2,500 g for 5 min at room temperature) and resuspended in 500 μL extraction buffer. Proteins were removed by digesting the samples with 20 μL of 20 mg/mL proteinase K (Applichem), 30 min at 55°C with shaking (1,000 rpm). Before an O/N incubation at 65°C with shaking (1,000 rpm), 130 μL of 5M sodium chloride were added.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-01-03T15:59:19Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-03-26T16:43:55Z
attributes.tag: External Id
attributes.value: SAMEA5537215
attributes.tag: INSDC center name
attributes.value: Max Planck Institute for Molecular Biomedicine
attributes.tag: INSDC first public
attributes.value: 2020-01-03T15:59:19Z
attributes.tag: INSDC last update
attributes.value: 2019-03-26T16:43:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7816:Scc1+Aux_Batch3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES-E14 cell
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: date of experiment
attributes.value: 11.06.2018
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: genotype
attributes.value: E14_Scc1_AID
attributes.tag: sample name
attributes.value: E-MTAB-7816:Scc1+Aux_Batch3
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: 129Ola
attributes.tag: treatment
attributes.value: Auxin treatment for 6 hours