title: GSM2454138: ChIP T; 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 LB1 (50mM Hepes pH7.5, 140mM NaCl, 1mM EDTA pH8, 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 with a tight pestle with 3x 15 strokes, with short resting times on ice in between. The chromatin was pelleted by centrifugation at 1400g and 4°C for 5 minutes and resuspended in 2.5ml LB2 (200mM NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 10mM Tris pH 8) per cell pellet. After 10 minutes rotation at 4°C, the centrifugation step was repeated and each pellet was resuspended in 1.5ml LB3 (1mM EDTA pH8, 0.5mM EGTA, 10mM Tris pH8, 100mM NaCl, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine), transferred to 15ml Falcon tubes and sonicated using a W-450D Digital Sonifier (Branson) sonicator for 14 cycles of 10s on/50s off in a 4°C coldroom with tubes chilled in ice water. After sonication, 150ul of triton X-100 was added per tube, transferred to two 1.5ml 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 50ul as an input control, the chromatin was distributed into 1.5ml aliquots, snap frozen and stored at -80C until use.  The input was reverse-crosslinked with the addition of 50ul 2x elution buffer (100mM Tris pH8, 20mM EDTA pH8, 2% SDS) and overnight (13h-15h) incubation at 65°C. The next day, 100ul of TE buffer were added to dilute the SDS of the elution buffer and RNA was digested using RNase A at a 0.2ug/ml final concentration at 37°C for 2 hours. Proteins were digested with the addition of 0.2ug/ml proteinase K and incubation at 55°C for 2 hours. The DNA was purified with two subsequent phenol:chloroform:isoamylalcohol (25:24:1, pH8) isolations and subsequent MinElute (Qiagen) purification. Purification was performed according to manufacturer’s recommendations, except that the columns were washed 2x with PE and air dried for at least 10 minutes before elution with EB. The input DNA was quantified using a NanoPhotometer (Implen) and the shearing was verified by running 600ng of DNA on a 1.5% agarose gel. For sonication to be considered successful and sonicated chromatin to be usable for ChIP, the bulk DNA of the input had to be located between 100bp-500bp.  For ChIP, the indicated amount of protein-coated Dynal beads (Life) was washed 3x using 1ml of blocking solution (PBS containing 0.5% BSA), resuspended in 500ul of blocking solution together with the antibody and rotated overnight at 4°C. The next day, three further washes using 1ml blocking buffer were performed and the beads were resuspended in 100ul blocking buffer. The indicated amount of chromatin was added and rotated overnight at 4°C. The following day, washing steps were performed using 1ml RIPA buffer (50mM Hepes pH7.6, 500mM LiCl, 1mM EDTA pH8, 1% NP-40, 0.7% Na-Deoxycholate) containing 1x complete protease inhibitors without EDTA and finally with 1ml of TEN (10mM Tris pH8, 1mM EDTA pH8, 50mM NaCl). The immunoprecipates were eluted in two subsequent steps using 100ul of 1x elution buffer (50mM Tris pH8, 10mM EDTA pH8, 1% SDS) and incubation at 65°C while shaking for 10 minutes each. The eluates were combined and incubated overnight (13h-15h) at 65°C under agitation. The next day, 200ul of TE were added to dilute the SDS of the elution buffer and the ChIPs were purified as described for the input above. ChIP DNA was quantified using the Qubit (Life) DNA HS assay. The ChIP-Seq libraries were generated using the TruSeq ChIP library kit (Illumina) with the following modifications. After adapter ligation, 0.95x of AMPure XP beads (Beckman Coulter) were used for a single purification and the DNA was eluted using 15ul of resuspension buffer (RSB, Illumina). Of the eluate, 14ul were used for PCR pre-amplification in order to convert adapters into double-stranded DNA. After the addition of 1ul primer mix (25mM each) and 15ul 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 21ul of RSB. After transfer of 20ul of eluate, 6ul of 5x loading buffer was added and the ligation products were separated using a 1.5% agarose gel. Post-run staining was performed using Sybr Gold (Life) under agitation for 30 minutes. Gel slices according to ~250-400bp fragment size were cut out using a Dark Reader (Clare Chemical Research) transilluminator. The gel extraction was performed using 5 gel volumes of QG buffer (Qiagen) with the addition of 1 gel volume of isopropanol, with each flow through added to the column a second time. 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 21ul of EB buffer. Of the eluate, 19ul was used in the final amplification, with the addition of 1ul primer mix and 20ul 2x Kapa HiFi Hotstart premix. The same protocol as for the pre-amplification was used, with the exception of using 13 cycles, resulting in a total amplification of 18 cycles. The libraries were quantified using the Qubit DNA HS assay and the library size was validated using DNA HS bioanalyzer chips.