GSM7060152: Input_SRRM2_TSAseq; Mus musculus; OTHER
1% formaldehyde was added to 7.5x10^6 mouse ESCs to cross-link proteins to DNA. Cells were then permeabilized in PBS with TritonX-100 0.5% for 5 min on ice and 10 min at RT. Next, cells were resuspended in 1.5% H2O2 to quench the endogenous peroxidases by slowly nutating at RT for 1 hr. After three washes with PBS-TritonX-100 0.1% (PBST), cells were resuspended in GS blocking buffer (1% BSA in PBST) for 1 hr at RT. Cells were then incubated with antibody anti-SRRM2 (Novus Biologicals, NBP2-55697) 1:500 in GS blocking buffer overnight at 4°C. For the no-antibody control sample, cells were incubated in GS blocking buffer only. After washing, cells were incubated with HRP conjugated secondary antibody (Vector Laboratories, PI-1000-1) 1:1000 in GS blocking buffer for 2 hr at RT. Cells were then incubated in Buffer A (50% sucrose, 0.0015% H2O2, 1:2500 tyramide-biotin 8 mM (Sigma-Aldrich, SML2135) for 10 min nutating at RT. For monitoring TSA labeling by immunostaining, a small aliquot of each sample was placed on a glass coverslip, air dried for few minutes and fixed on the coverslip with 3.7% formaldehyde in PBS for 10 min at RT. After washing in PBST three times, coverslips were incubated 30 min at RT with streptavidin-Alex Fluor 594 (Invitrogen, S11227) 1:1000 and Alex Fluor 488 secondary antibody (Thermo Fisher, A11034) 1:1000 in GS blocking buffer. After washing, the coverslips were mounted using VECTASHIELD® Antifade Mounting Medium with DAPI. To shear the DNA, cells were resuspended in MNase digestion buffer (0.3M Sucrose, 50mM Tris pH 7.5, 30mM KCl, 7.5mM NaCl, 4mM MgCl2, 1mM CaCl2, 0.125% NP-40, 0.25% NaDeoxycholate, supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche)) with 100U MNase (Roche) at 37°C for 30 minutes. The digestion was then stopped with 5mM EDTA and the digested chromatin was solubilized in 200mM NaCl and five pulses of 30sec sonication using a Bioruptor® Pico ultrasonic cell disruptor (Diagenode). For the affinity purification of biotinylated DNA, 10μl/106 cells of Dynabeads™ M-270 streptadivin (Invitrogen) were washed with Wash and Binding (W&B) buffer (5mM Tris pH 7.5, 0.5 mM EDTA, 1M NaCl) before being added to the samples. Beads were then washed four times with W&B buffer, with TSE 500 (20 mM Tris pH 8, 1% TritonX-100, 0.1% SDS, 2 mM EDTA pH 8, 500 mM NaCl), again with W&B buffer and then with TE buffer. After the final wash, beads were resuspended in Proteinase K buffer (10 mM Tris pH 7,5, 10 mM EDTA pH 8, 0.5% SDS) supplemented with free biotin 0.1 mM and 40 μg of Proteinase K (Thermo Scientific) and incubated at 50°C for 3 h and reverse cross-linked at 65°C, overnight. DNA was isolated by Phenol:Chloroform extraction following original protocol's instructions. Quantity and quality of the isolated DNA was determined with Qubit® 4 Fluorometer (Life Technologies). The NEBNext® Ultra™ II DNA Library Prep for Illumina (New England Biolabs, E7645S and E7645L) was used following the manufacturer's protocol. Briefly, TSA and input samples (10 ng) were end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® 4 Fluorometer and the 4200 TapeStation System (Agilent). Sequencing was performed on an Illumina NovaSeq6000 machine with single-end 100 bp reads.