title: GSM2401551: CV_Dnase_3; Mus musculus; DNase-Hypersensitivity
library_construction_protocol: Mice were euthanized under CO2 and cervical dislocation and placed on a chilled wax dissection pad.  The small intestine was removed from the mouse and the jejunum was excised from the duodenum and ileum (Duodenum was defined as the anterior 5 cm of the midgut and ileum was defined as posterior 6 cm of midgut as described (Camp, et al 2014).  Adipose and vasculature were removed from the tissue.  The jejunum was opened longitudinally along the length of the tissue, exposing the lumen and epithelial cell layer.  Luminal debris was washed away from the epithelia with ice cold sterile PBS.  The tissue was temporarily stored in 10 ml of ice cold sterile PBS with 1x Protease Inhibitor (Complete EDTA-Free, Roche 1187350001) and 10 uM Y-27632 (ROCK I inhibitor, Selleck Chemicals S1049) to inhibit spontaneous apoptosis.  The jejunum was moved into a 15 ml conical tube containing 3 mM EDTA in PBS with 1x protease inhibitor and 10 uM Y-27632.  The tissue was placed on a nutator in a cold room for 15 minutes.  The jejunum was removed from the 3 mM EDTA and placed on an ice cold glass petri dish with PBS containing 1mM MgCl2 and 2 mM CaCl2 with protease inhibitors and 10 uM Y-27632.  Villi were scraped off of the tissue using a sterile plastic micropipette and placed into a new 15 ml conical tube.  The isolated IECs were then pelleted at 250 x g at 4°C for 5 minutes, resuspended in 15 ml of ice cold PBS containting 10 uM Y-27632 and 1x protease inhibitors and pelleted again at 250 x g at 4°C.  The cell pellet was used for chromatin immunoprecipitation or for nuclear extractions. [ChIP-seq] Frozen sonicated chromatin was thawed on ice.  Thawed and fresh chromatin samples were diluted in 1 mL of ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl (pH 8.1), and 150 mM NaCl) containing 1x protease inhibitor and precleared with washed protein G Dynabeads for 3 hours at 4°C on a nutator.  Precleared chromatin was incubated with ChIP grade antibodies [4 ug H3K4me1 (Rabbit anti-H3K4me1, Abcam ab8895), 4 ug H3K27ac (Rabbit anti-H3K27ac, Abcam ab4729), 8 ug Hnf4a (Mouse anti-Hnf4a, Abcam 41898), 8 ug Hnf4g (Goat anti-Hnf4g, Santa Cruz sc-6558X), 8 ug CTCF (Rabbit anti-CTCF, Active Motif, 61311)] overnight at 4°C on a nutator.  Antibody-chromatin complexes were pulled down with washed protein G dynabeads for 4 hours at 4°C on a nutator.  The beads were washed 5x for 3 minutes with ice cold LiCl wash buffer (100 mM Tris-Cl (pH 7.5), 500 mM LiCl, 1% IGEPAL, 1% sodium deoxycholate)) and 1x with ice cold TE buffer at 4°C on a nutator.  Washed beads were resuspended in 100 uL of ChIP elution buffer (1% SDS and 0.1 M sodium bicarbonate)) and placed in a thermomixer heated to 65°C and programed to vortex at 2000 RPM for 15 seconds, rest for 2 minutes for a total of 30 minutes.  The beads were pelleted, placed on a magnet, and the supernatant was moved to a new tube.  This elution process was repeated once and corresponding elutions were combined for a total of 200 uL.  To reverse crosslink immunoprecipitated chromatin, 8 uL of 5 M NaCl was added to each 200 uL ChIP elution and elutions were incubated at 65°C overnight.  Immunoprecipitated chromatin was isolated using a QIAquick PCR quick preparation kit (Qiagen 28104), quantified using a Qubit 2.0 flourometer and stored at -80°C until library preparations and amplification.  Libraries were always prepared within 3 days of the immunoprecipitation with the NEBNextUltra DNA Library Prep Kit for Illumina (New England Biolabs E7370S).  Prepared libraries were quantified using a Qubit 2.0 fluorometer and submitted to Hudson Alpha Genomic Services Laboratory for 50 bp single end sequencing on an Illumina HiSeq 2500 with 4 samples per lane in the flow cell. [RNA-seq] Mouse jejunum intestinal epithelial cells were collected as mentioned above.  Prior to crosslinking, 1/50 of the isolated IECs were suspended in 1 ml TRIzol and stored at -80°C.  200 uL of chloroform was added to the TRIzol and the sample was vortexed on high for 30 seconds at room temperature.  The samples were incubated at room temperature for 2 minutes and centrifuged at 12,000 x g for 15 minutes at 4°C.  The top aqueous layer was removed and added to equal volume of isopropanol.  The nucleic acids were isolated using a column-based RNA-isolation kit (Ambion Cat 12183018A) with an on column DNase I (RNase-free) treatment (New England Biolabs M0303L) to remove DNA contamination.  RNA was eluted off the column in nuclease-free water, quantified using a Qubit 2.0 and stored at -80°C until submission to the Duke Sequencing and Genomic Technologies Core.  RNA-seq libraries were prepared and sequenced by Duke Sequencing and Genomic Technologies Core on an Illumina HiSeq 2500 with 4 samples per lane in the flow cell. [Dnase-seq] DNase hypersensitivity was performed as described (Camp et al., 2014) with the following modifications: IECs were isolated as above from jejunum and subjected to endogenous DNase activity to digest chromatin.  DNase-seq libraries were constructed as previously described, with “Oligo 1b” phosphorylated at the 5’-end to enhance ligation efficiency (Song and Crawford, 2010).  Libraries were sequenced by Illumina HiSeq 2000 with 50 bp single end reads with 3 samples per lane. [Input] Germ free or conventionalized chromatin for input normalization was generated using the same protocol as above except no antibody was used during the overnight antibody incubation; instead, chromatin was incubated at 4°C with gentle agitation.  Bead incubation, reverse-crosslinking and library preparations for these samples were performed using the same protocol
identifiers.namespace: GEO
identifiers.id: GSM2401551