title: GSM6663203: ChIP Control P-Smad1/5/9 Day2; Mus musculus; ChIP-Seq
library_construction_protocol: For sonication, complete protease inhibitors without EDTA (Roche) at 1x final concentration was added to all lysis buffers (LB) prior to use. Each pellet was resuspended in 2.5 ml LB1 (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.75% NP-40, 0.25% triton X-100) and rotated at 4°C for 20 minutes. The cell suspension was homogenized using a douncer. The chromatin was pelleted by centrifugation at 1400g and 4°C for 5 minutes and resuspended in 2.5 ml LB2 (10 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). After 10 minutes rotation at 4°C, the centrifugation step was repeated and each pellet was resuspended in 1.5 ml LB3 (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine), transferred to 15 ml Falcon tubes and sonicated using a W-450D Digital Sonifier (Branson) sonicator for 14 cycles of 10s on/50s off in a 4°C cold room with tubes chilled in ice water. After sonication, 150 μl of Triton X-100 was added per tube, transferred to two 1.5 ml Eppendorf tubes and debris was pelleted by centrifugation at 20,000g and 4°C for 10 minutes. The solubilized chromatin was then pooled and mixed thoroughly. After taking 50 μl as an input control, the chromatin was distributed into 1.5 ml aliquots, snap frozen and stored at -80°C until use.  The input was reverse-crosslinked with the addition of 50 μl 2x elution buffer (100 mM Tris pH 8, 20 mM EDTA pH 8, 2% SDS) and 13-15 hour incubation at 65°C. 100μl of TE buffer were added, and RNA was digested using 0.2 μg/ml RNase A at 37°C for 2 hours. Proteins were digested with the addition of 0.2 μg/ml proteinase K and incubation at 55°C for 2 hours. The DNA was purified with two subsequent phenol:chloroform:isoamyl alcohol (25:24:1, pH 8) extractions and a subsequent MinElute (Qiagen) purification. The input DNA was quantified using a NanoPhotometer (Implen).    For ChIP, 100 μl protein G-coated Dynal beads (Life Technologies) were washed 3x with 1 ml of blocking buffer (PBS, 0.5% BSA), resuspended in 500 μl of blocking solution containing 5 μg primary ChIP antibody (listed in the Key resources table) and rotated overnight at 4°C. Beads were then washed 3x with 1 ml of blocking buffer and resuspended in 100 μl blocking buffer. Chromatin equivalent to 5x107 cells was added and rotated overnight at 4°C. The following day, 6 washing steps (9 for T) with 1 ml RIPA buffer (50 mM Hepes pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and one washing step with 1 ml of TEN (10 mM Tris pH 8, 1 mM EDTA, 50 mM NaCl) were performed. The elution was performed in two subsequent steps using 100 μl of 1x elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) and incubation at 65°C while shaking for 10 minutes each. The eluates were combined and incubated for 13-15 hours at 65°C. 200 μl of TE were added and the ChIP DNA was purified as described for the input above. ChIP DNA was quantified using the Qubit (Life Technologies) DNA HS assay. The ChIP-seq libraries were generated using the TruSeq ChIP library kit (Illumina) with the previously described modifications (Koch, 2017). After adapter ligation, 0.95x of AMPure XP beads (Beckman Coulter) were used for a single purification and the DNA was eluted using 14 μl of resuspension buffer (RSB, Illumina). After the addition of 1 μl primer mix (25 mM each) and 15 μl 2x Kapa HiFi HotStart Ready Mix (Kapa Biosystems), amplification was performed for 45 seconds at 98°C, 5 cycles of [15 seconds at 98°C, 30 seconds at 63°C and 30 seconds at 72°C] and a final 1 minute incubation at 72°C. The PCR products were purified using 0.95x of beads and eluted using 21 μl of RSB. Ligation products were then separated using a 1.5% agarose gel. Post-run staining was performed using SYBR Gold under agitation for 30 minutes. Gel slices corresponding to 250-400 bp fragment size were cut out. using a Dark Reader Transilluminator.  The gel extraction was performed using 5 gel volumes of QG buffer (Qiagen) with the addition of one gel volume of isopropanol. The MinElute (Qiagen) columns were washed once with QG buffer and twice with PE buffer, air-dried for at least 10 minutes and eluted using 21 μl of EB buffer. 19 μl of the eluate was used in the final amplification, with the addition of 1 μl primer mix and 20 μl 2x Kapa HiFi Hotstart premix. The same protocol as for the pre-amplification was used, with the exception of using 13 amplification cycles. The libraries were quantified using the Qubit DNA HS assay and the library size was validated using DNA HS Bioanalyzer chips.