title: P21_H3K4me1_2
organism: Mus musculus
description: Protocols: MATERIAL COLLECTION. Intestinal epithelial cell isolation are based on protocol by Sato and Clevers, 2013. The proximal half of mouse small intestine was isolated and washed with ice-cold PBS until luminal contents were cleared. The intestine were opened longitudinally cut into ~2mm pieces. These fragments were washed 5 times with ice cold PBS. Tissue pieces were incubated with 2 mM EDTA in ice-cold PBS for 30-60 minutes and cells from 2-3 mice where pooled. Intestinal epithelium cells were isolated from up to 10 fractions after pipetting up and down 5 times with PBS. To isolate single cells for sorting, IECs were incubated in TrypLE (ThermoFisher) at 37 °C for 20-45 minutes.CELL ISOLATION. Dapi-negative, Epcam-positive IECs were sorted in PBS containing 20 mM Sodium Butyrate, and cross-linked by incubation in 1% formaldehyde for 8 minutes. Glycine was added to a final concentration of 125 mM and incubated for 5 minutes at room temperature. Using a swing-out rotor cells were washed 3 times in ice-cold PBS with 20 mM Sodium Butyrate. After washing, cells were snap frozen in liquid nitrogen and stored in -80 The ChIP-seq was carried out similarly to previously described protocols(Dahl and Collas, 2008). BINDING OF ANTIBODIES TO PARAMAGNETIC BEADS. The stock of paramagnetic Dynabeads Protein A was vortexed thoroughly to ensure a homogenous suspension before pipetting. Dynabeads stock solution (5 μL per IP) was transferred into a 1.5-ml tube, which was placed on a magnetic rack and the beads captured on the tube wall. The buffer was discarded, the beads washed twice with 200 μL standard RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and resuspended in standard RIPA buffer to a final volume of 100 uL per IP. 99 μL of this was aliquoted into each 0.6 mL tube on ice, and antibody (1.2 ug of anti-H3K4me1: Diagenode, C15410194, Lot A1862D or 4 uL anti-H3K4me2: Cell Signaling #9725, Lot 9) was added per 0.6 mL-tube. Tubes were then incubated at 40 r.p.m. on a 'head-over-tail' tube rotator for at least 16h at 4 °C. CHROMATIN PREPARATION. Crosslinked cell pellets containing 50 000-300 000 cells were thawed on ice. The 10 μL pellets were added a modified Lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.8% SDS, 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail) to a total of 120μL and incubated on ice for 10 min, followed by addition of 30 uL PBS with 20 mM sodium butyrate. The samples were sonicated as described above. After the final sonication, 360 uL RIPA without SDS (with 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail) was added to the tube while washing the probe, followed by thorough mixing by pipetting. The sample and input were centrifuged at 12,000g in a swinging-bucket rotor for 10 min at 4 °C and the supernatants were transferred to a 1.5 ml-tube on ice. Chromatin corresponding to 1 000 - 2 000 cells was removed from each sample to be decross-linked and sequenced as inputs. For the ChIPs, chromatin corresponding to 50 000 cells was used for E18.5 Replicate A, while chromatin from 100 000 cells was used for the remaining samples. IMMUNOPRECIPITATION AND WASHES.Pre-incubated antibody-bead complexes were washed twice in 200 μl standard RIPA buffer by vortexing roughly. The tubes were centrifuged in a mini-centrifuge to bring down any solution trapped in the lid and antibody-bead complexes were captured in a magnetic rack. After removal of RIPA, 177-500 μl of chromatin (equivalent of 50,000-100,000 cells per ChIP) was added to each tube, then incubated at 4 °C, 40 r.p.m. on a 'head-over-tail' rotator for at least 16 h. For H3K4me1 ChIPs, the chromatin- antibody-bead complexes were washed three times in 100 μl ice-cold standard RIPA buffer. For H3K4me2 ChIPs, the reactions were washed once in standard RIPA, once in RIPA with increased salt and SDS (300 mM NaCl and 0.20% SDS), once in RIPA with increased salt and SDS (300 mM NaCl and 0.23% SDS), once in standard RIPA. All washing steps were performed with 100uL ice cold buffer supplemented with 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail. Each wash involved rough vortexing at full speed, repeated twice with pauses on ice in between. Next, a wash in 100μl TE and tube shift was carried out. REFERENCES. Sato, T., Clevers, H., 2013. Primary mouse small intestinal epithelial cell cultures. Methods Mol. Biol. 945, 319-328. doi:10.1007/978-1-62703-125-7_19Dahl, J.A., Collas, P., 2008. A rapid micro chromatin immunoprecipitation assay (ChIP). Nat Protoc 3, 1032-1045. doi:10.1038/nprot.2008.68 DNA ISOLATION AND PURIFICATION. TE was removed and 150μl ChIP elution buffer was added (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA. 1% SDS, 30 μg RNase A) and incubated at 37 °C, 1 h at 1,200 r.p.m. on a Thermomixer. The input samples were added ChIP elution buffer up to 150uL and incubated similarly. 1μl of Proteinase K (20 mg/ml stock) was added to each ChIP or input tube and incubated at 68 °C, 4 h at 1,250 r.p.m. The ChIP eluates were transferred to a 1.5-ml tube. Then, a second elution with 150μl was performed for 5 min and pooled with the first supernatant. The ChIP and input DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated with 11μl acrylamide carrier and dissolved in 10-15μl EB (10 mM Tris-HCl). ChIP and input library preparations were performed according to the QIAseq Ultralow Input Library Kit procedure.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-11-16T09:25:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-21T20:27:48Z
attributes.tag: External Id
attributes.value: SAMEA6495614
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2020-11-16T09:25:14Z
attributes.tag: INSDC last update
attributes.value: 2020-01-21T20:27:48Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8710:P21_H3K4me1_2
attributes.tag: age
attributes.value: 21
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: juvenile
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: F
attributes.tag: organism part
attributes.value: epithelium of small intestine
attributes.tag: sample name
attributes.value: E-MTAB-8710:P21_H3K4me1_2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL/6