title: GSM3381974: DE SMAD2/3 Chip (2); Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde (Sigma, F1635) at 37°C for 15 min and quenched with 0.125 M glycine for 5 minutes at room temperature. Fixed cells were scraped off the plates in cold PBS buffer and washed twice in cold PBS buffer. Cell pellets were obtained by centrifugation at 3000 RPM for 5 minutes at 4 oC and frozen in liquid nitrogen immediately before transferring to the -80C freezer.  ~25 million cells were used for one ChIP reaction. Cell pellets were thawed on ice, resuspended in 1ml SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8) containing proteinase and phosphatase inhibitor for one reaction in an eppendorf tube and incubated on ice for 10 minutes. Sonication was performed on a Branson Sonifier 250 with a 20% amplitude setting for 5.5 minutes (10 seconds on/off pulsing). Sonication products were spun down at 14000 RPM at 4oC for 10 minutes and 1ml supernatant containing chromatin and DNA were transferred to a falcon tube containing 9ml ChIP dilution buffer (0.01% SDS, 1.1% TritonX-100, 1.2mM EDTA, 16.7 mM Tris-HCl pH8, 167 mM NaCl) with proteinase and phosphatase inhibitor. 50 μl of dynabeads (Life Technologies 10009D) were added to samples and incubated at 4oC with rotation for 1 hour. After pre-clearing, dynabeads beads were removed and 200 μl of sample were collected as 2% input separately. 5 μg antibodies were added to the pre-cleared samples for overnight incubation at 4oC with rotation. 200 μl dynabeads were added into one ChIP reaction and incubated for 4-6 hours at 4 oC with rotation.  Dynabeads were collected by centrifugation with 3000 RPM at 4 oC for 5 minutes and washed in 1ml low salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 150 mM NaCl) for 5 minutes at 4oC with rotations. Then beads were washed in 1ml high salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 500 mM NaCl) twice and TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA) twice for 5 minutes at 4oC with rotation. After the last wash, beads were resuspended in 250 μl elution buffer (1% SDS, 0.1 M NaHCO3) and incubated in a thermomixer (850RPM) for 15 minutes at 60 oC. Supernatant were collected and added with 5 M NaCl for overnight decrosslinking at 65 oC. 10 μl 0.5 M EDTA, 20 μl 1 M Tris-HCl pH6.5 and 1 μl proteinase K (20 mg/ml) were added to decrosslinked product and incubated for 1 hour at 45 oC. DNA were isolated by using QIAquick PCR purification kit (Qiagen 28104). Libraries were prepared using the NEBNext® ChIP-seq Library Prep Master Mix Set for Illumina ® (NEB, E6240L) and quality controlled using Agilent Technologies 2200 TapeStation to determine fragment size and PicoGreen (Life Technologies/Invitrogen, P7589) to quantify the concentration. Samples were pooled and submitted to New York Genome Center for SE50 sequencing using a HiSeq 2500.
identifiers.namespace: GEO
identifiers.id: GSM3381974