GSM4460174: Mat-IGTRE-del_Gtl2-VP_rep2; Mus musculus; Mus musculus molossinus; OTHER
Cells were collected with trypsin and fixed with 1% formaldehyde Cell pellets were lysed in 1ml Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA; 1x complete protease inhibitor, 0.5% NP-40, 1% triton) and incubated on ice for 15 min. The samples were centrifuged at 2500xG for 5min at 4°C and the pellet was then resuspended in 360µl milli-Q, 60µl 10X DpnII restriction buffer and 15ul 10%SDS. After 1 hour incubation at 37ºC, 150ul of 10% Triton was added and samples were incubated again at 37ºC for 1 hour. 4ul DpnII enzyme (#R0543M, NEB) were added and samples were incubated at 37ºC over night while shaking in a thermomixer (9000rpm). After confirming the digestion efficiency, the enzyme was inactivated by adding 80ul 10% SDS and incubating at 65 ºC for 30 mins. The digested samples were then diluted with 4860ul Milli-Q, 700ul ligation buffer (500mM Tris pH 7.5, 100mM DTT, 100mM MgCl2,10mM ATP), and 750ul of Triton and incubated at 37ºC for 1 hour. Then 2ul Ligase (NEB M0202M) were added and samples were incubated over night at 16 ºC. Next morning, after testing the ligation efficiency, we reversed the crosslinks by adding 30ul of proteinase K (10mg/ml) and incubating over night at 65ºC. Subsequently the RNA was removed using 30ul of RNase A (10mg/ml) for 45mins at 37ºC. Extensive phenol/chloroform extraction was followed by EtOH precipitation and two washes with 70% EtOH. The pellets were dissolved in 150ul 10mM Tris pH 7.5 by incubating at 37 ºC. We then added 50ul 10x buffer B (Fermentas), 5ul Csp6I (Fermentas, ER0211) and 299ul milli-Q water and samples were digested at 37ºC over night. After determining digestion efficiency, the restriction enzyme was inactivated by incubating the tubes at 65ºC for 25 mins. Samples were diluted in 12ml milli-Q, 3ul ligase (NEB, M0202M) and 1.4ml 10X ligation buffer (500mM Tris pH7.5, 100mM DTT, 100mM MgCl2, 10mMATP) and incubated over night at 65ºC. Following phenol/chloroform and EtOH precipitation the pellets were dissolved in 300ul 10mM Tris pH7.5 and DNA was further purified using 4 Zymo columns per sample (Zymo, D4014). Each sample was eluted in 200ul total of 10mM Tris pH7.5 For allele-specific 4C-seq library preparation, primers were designed upstream of a SNP within IG-DMR. Due to lack of suitable restriction fragments on ΔMatTRE samples, non-allele specific libraries were prepared with the Gtl2 DMR as viewpoint. Library preparation was further performed by using a PCR strategy as previously described (Krijger et al., 2020). Briefly, 4x200 ng of 4C-template DNA was used to PCR amplify the libraries using the Roche Expand long template PCR system (Roche, 11681842001). Primers were removed using Ampure XP beads (Beckman Coulter, A63880). A second round of PCR was performed using the initial PCR library as a template, with overlapping primers to add the full adapters. The libraries were sequenced on a miSeq platform in SE150 mode.
GEO Accession
GSM4460174