title: GSM4684629: UNDIFF6-ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Use of two independent biological replicates for ChIP-seq analysis to identify transcriptional targets is consistent with ENCODE (Encyclopedia of DNA Elements) guidelines. For ChIP-seq studies,  four independent ChIP experiments were performed, two with undifferentiated cultures and two for differentiated cultures, to enable analysis of the effect of differentiation on BMAL1 targets. Briefly, undifferentiated and differentiated HC11 cultures in 100 mm plates were treated with 1% formaldehyde to cross-link proteins to chromatin by adding 625 µl fresh 16% formaldehyde directly to the dishes containing cells and 10 mL media, and incubated at RT on a shaking platform for 7 min. To quench the formaldehyde, 500 µl of 2.5M glycine was added, and incubated at RT on the shaking platform for 5 min. Cells were washed twice with ice cold PBS, and removed from culture dish using a cell scraper.  Cells were pelleted by centrifugation and stored at the -80 °C. Samples were collected for ChIP assays in four replicate experiments (sample IDs UNDIFF6, UNDIFF8, DIFF1, DIFF2).    Nuclei were collected by resuspending each fixed-cell pellet in 10 mL of Rinse 1 (50mM Hepes pH 8.0, 140mM NaCl, 1mM EDTA, 10% glycerol, .5% NP40, .25% Triton x100), and incubating 10 min on ice. Samples were pelleted by centrifugation 1,200 X g at 4 °C for 5 min, and then resuspend in 10 mL CiA NP-Rinse 2 (10mM Tris pH 8.0, 1mM EDTA, 0.5mM EGTA, 200mM NaCl), and then centrifuged at 1,200 X g and 4 °C for 5 min.  Pellets were washed with 5 ml of CiA Covaris Shearing Buffer (0.1% SDS, 1mM EDTA pH 8.0, 10mM Tris HC1 pH 8.0) twice with centrifugation at 1,200 X g and 4 °C for 3 min after each wash. For sonication, pellets from approximately 3 million cells each were resuspended in 130 µl ofCiA Covaris Shearing Buffer with 10% protease inhibitor cocktail.  Pellets were sonicated in a Covaris E210 instrument (Covaris, Inc., Woburn, MA) using the following parameters: Duty Factor 5%, Peak power 105W, Cycles per burst 200, 10 min.  Following sonication sheared chromatin lysate was transferred to a new centrifuged at 20,000 X g and 4 °C for 15 min.  Supernatant (sheared chromatin stock) was divided into three aliquots for input DNA, mock-immunoprecipitation (IP) and IP and stored at -80 °C (-20C), after removal of an aliquot for analysis of product size in a 2% agarose gel and on the Agilent Bioanalyzer Nanochip. For immunoprecipitation step, cross-linked chromatin using anti-BMAL1 antibody and mock-IP aliquots were thawed on ice.  Samples were precleared by adding 50 µL resuspended A beads (DynaBeads Protein A, Invitrogen cat. # 10001D), and incubating at 4° C for 1.5 h with rotation.  Tubes were placed on a magnetic rack and liquid was transferred to a new microfuge tube.  One-quarter of the volume of 5X IP Buffer (250mM Hepes/KOH pH 7.5, 1.5M NaCl, 5mM EDTA, 5% TritonX 100, 0.5% DOC [4-chloro-2,5-dimethoxy-amphetamine], 0.5% SDS) was added, and 10 µL of BMAL1 antibody (Abcam ab 3350) was added to IP tube, and nothing was added to mock-IP tube.  Samples were rotated overnight at 4° C, and then 50 µL of resuspended A beads were added to each tube, and rotated for 3 h at 4° C.  Magnetic rack was used to remove A beads. Supernatant was collected transferred to a new tube, and washed with 1mL IP Buffer twice with rotation at RT for 3 min, and collection on magnetic rack following wash.  Supernatants were then washed with 1 mL of DOC buffer (10 mM Tris, pH8.0; 0.25 M liCl; 0.5% NP40, 0.5% DOC, 1 mM EDTA), followed by 1 mL of Tris EDTA (TE) buffer pH 8. Supernatant was removed and 150 µL elution buffer was added and samples were rotated at RT for 20 min. Supernatant was transferred to a new tube and store at -80 °C until DNA isolation. Chromatin was isolated from supernatant by adding 3 volumes of TE with 1% SDS and RNAse A (Sigma R6513), and vortexing to mix. Samples were incubated at 37° C for 30 min, and proteinase K was added, vortexed again to mix, and cross-linking was reversed by incubating at 55° C for ~2.5 h, and then overnight at 65° C in PCR tubes in the thermocycler.  DNA was extracted the next day by adding 230 µL TE and 330 µL phenol/chloroform, vortexing, followed by centrifugation for 1 min.  Aqueous layer was transferred into a new microfuge tube and 10 µL of commercial glycogen (ThermoScientific R0551) was added with 30 µL 3M NaCl.  One volume of 100% ethanol was added and samples vortexed to mix. Samples were incubated for two days at -20 ° C, and then centrifuged 1 h at 4 °C to pellet DNA. Pellet was washed twice by removing the supernatant and adding 500 µl 70% EtOH. Pellet was resuspended in 25 µL TE and concentration was measured with Qubit fluorometer (Thermo Fisher Scientific) and a Bioanalyzer 2100 (Agilent), and samples stored at -80° C until sequencing. Libraries were prepared using NEXTFLEX® Rapid DNA-Seq Library Prep Kit for Illumina® Platforms (PerkinElmer, ) according to manufacturer's protocol. Libraries were sequenced by paired ends reads on the Illumina Novaseq 6000 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM4684629