GSM9267176: FKBP-SETDB1 P2F7 ES, dTAG+dox, RNAPolII; Mus musculus; ChIP-Seq
For RNA polymerase II calibrated ChIP-seq (cChIP-seq), 5x107 mESCs were pooled with 1x107 Drosophila cells after collection, washed in PBS wash and counted using a Countess II FL Automated Cell Counter (Life Technologies). Cells were crosslinked for 10min with 1% formaldehyde. The reaction was quenched using glycine for additional 10min before recovering the cells by centrifugation at 1000g for 5min at 4oC. After a PBS wash, supernatant was removed and cells were snap frozen and stored at -80oC until use. Cell pellets were thawed on ice, resuspended in 1mL of FA-Lysis Buffer (50 mM HEPES pH7.9, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1%  Sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors, 1 mM AEBSF, 10 mM NaF and incubated on ice for 10min prior to sonication using a Bioruptor Pico Sonicator (Diagenode) for 30 cycles (30sec on/off). Post-sonication chromatin was spun at maximum speed for 10min at 4oC and the supernatant was transferred to a new 1.5mL tube. For each IP, 300µg of chromatin were diluted to a total volume of 1mL in FA-Lysis Buffer and precleared by incubation with 40µL of pre-blocked rProtein A Sepharose™ Fast Flow bead slurry (Amersham) for 1h at 4oC with rotation. Pre-cleared chromatin was aliquoted in 1.5mL Protein lobind tubes and incubated overnight at 4oC with rotation with the corresponding antibody (15µL Anti-Rbp1 NTD, D8L4Y, Cell Signaling Technologies; 12.5µL Anti-Rbp1 Ser5P, D9N5I, Cell Signaling Technologies; 12.5µL Anti-Rbp1 Ser2P, E1Z3G, Cell Signaling Technologies). 10% of pre-cleared chromatin was kept as input. After incubation, 40µL of pre-blocked rProtein A Sepharose™ Fast Flow bead slurry (Amersham) were added to each sample and incubated for a minimum of 3h at 4oC followed by single 5min washes at 4oC with FA-Lysis, FA-Lysis 500 (same composition as FA-Lysis Buffer but with 500mM NaCl) and DOC (10 mM Tris pH8.0, 250 mM LiCl, 2 mM EDTA, 0.5% NP-40, 0.5% Sodium deoxycholate) buffers and two washes with TE pH8.0. The DNA was then eluted from the beads by incubation at 30oC for 30 min shaking at 1000rpm with 1% SDS, 100 mM NaHCO3 elution buffer and separated from the beads by centrifugation. IP and input samples were reverse crosslinked overnight and RNAse- and Proteinase K-treated, and the DNA was further purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research) according to manufacturer's instructions. For KAP1 and MPP8 ChIP-seq, 5x107 mESC cells were collected in PBS and double crosslinked by incubating with 2mM DSG for 50min at room temperature followed by an additional 12min with 1% formaldehyde. The reaction was quenched by addition of glycine and further incubation with rotation for 10min. Cells were pelleted by centrifugation, washed once with PBS and the pellet was snap frozen and stored at -80oC. To prepare chromatin, the cell pellets were thawed on ice and lysed in 10mL of LB1 (50mM HEPES-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100) supplemented with protease inhibitors for 10min, rotating at 4oC. Nuclei were recovered by centrifugation and resuspended in 10mL LB2 (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) with protease inhibitors and incubated rotating for 10min. After centrifugation, the pellet was resuspended in 1mL of LB3 (10mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine) with protease inhibitors per 5x 107 cells and subjected to sonication using a Bioruptor Pico (Diagenode) for 30 cycles (30 sec on/off). After sonication, samples were spun for 2 min 2000rpm at 4oC and the supernatant was collected into a new tube to which 110µL of LB3 with 10% Triton X-100 were added and mixed by inversion. Chromatin was spun at maximum speed for 10 min and the supernatant was collected into a new tube, snap frozen and stored. For immunoprecipitation, chromatin was thawed on ice and 110µL were taken per reaction and diluted in 990µL ChIP dilution buffer (1% Triton X-100, 1mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl). Chromatin was precleared by incubation with pre-blocked rProtein A Sepharose™ Fast Flow beads (Amersham) for 1 hour (40μL bead slurry/mL of chromatin) at 4oC and 100μL set aside as input. Antibodies (KAP1, Abcam, ab22553, 5µg; MPP8, Proteintech, 16796-1-AP, 7µg) were added to 1mL of precleared chromatin each and incubated overnight at 4oC with rotation. Samples were then incubated for 4h with 40µL of pre-blocked rProtein A Sepharose™ Fast Flow beads (Amersham) followed by single 4min washes with Low Salt (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), High Salt (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl), LiCl (0.25M LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA, 10mM Tris pH 8.0) wash buffers and two washes with 1xTE. The DNA was then eluted from the beads by incubation at 25oC for 30 min shaking at 1000rpm with 1% SDS, 100 mM NaHCO3 elution buffer and separated from the beads by centrifugation. IP and input samples were reverse crosslinked overnight and RNAse- and Proteinase K-treated, and the DNA was further purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research) according to manufacturer's instructions. ChIP and input DNA was quantified using the Qubit High Sensitivity DNA kit (Invitrogen) and submitted to library preparation using the the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) according to the manufacturer's instructions. Libraries were quantified using the Qubit High Sensitivity DNA kit (Invitrogen) and their length was estimated using the Bioanalyzer High Sensitivity DNA kit (Agilent) before pooling and subjected to paired-end sequencing on an Illumina NextSeq 500 machine using NextSeq 500/550 High-Output v2.5 Kit (150 cycles) (Illumina).