GSM8390203: MOF Degron mESC: Auxin-NascentRNA_TTSeq_Rep3; Mus musculus; OTHER
ChIP-Seq: ESCs were crosslinked with 1 % Formaldehyde in 1X PBS for 10 minutes at RT followed by quenching with 125 mM Glycine at 4 °C. After washing the ESCs twice with PBS, ESCs were lysed in LB1 buffer (50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10 % Glycerol, 0.5 % NP-40, 0.25 % Triton X-100) for 20 minutes at 4 °C. Then, samples were washed in LB2 buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 5 minutes at 4 °C. Chromatin was extracted by incubation in LB3 buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % DoC-Na, 0.5 % Na-Sarcosinate, 1 % Triton X-100) for 10 minutes on ice followed by sonication for 12 minutes on a Covaris sonicator. The sonicated chromatin was spun down at 16,000g for 10 minutes at 4 °C and the supernatant was incubated with the appropriate antibody overnight at 4 °C. Antibody-bound chromatin fragments were collected by incubation with Protein A or G DynaBeads for 3 h at 4 °C. The beads were washed for 5 minutes at 4 °C with the following buffers: RIPA-150 (50 mM Tris- HCl, pH 8.0, 150 mM NaCl, 0.1 % SDS, 0.5 % DoC-Na, 1 % NP-40), RIPA-500 (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1 % SDS, 1 % NP-40), Li-Buffer (50 mM Tris- HCl, pH 8.0, 250 mM LiCl, 0.5 % DoC-Na, 1 % NP-40), TE Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA). After an additional wash with TE Buffer, beads were resuspended in 100 μL of elution buffer (50 mM Tris-HCl, pH 7.4, 1 % SDS, 1 mM EDTA) and crosslinks were reversed by incubation at 65 °C for at least 4 h. Proteins and RNA were degraded by further incubations with RNase A and Proteinase K, respectively. Resulting DNA was purified using ChIP DNA Clean and Concentrator kit (Zymo) according to the manufacturer's protocol.                                                                                                                                                  Native Histone ChIP-Seq: 3 x 106 ESCs per replicate were harvested using accutase, washed once in 1X PBS and flash-frozen. The cell pellet was thawn on ice and resuspended in 150 μL of 1X PBS and mixed with 150 μL of 2X Lysis buffer (100 mM Tris-HCl, pH 8.0, 0.2 % Triton X-100, 0.1 % DOC-Na, 10 mM CaCl2, 10 mM Na-Butyrate) containing 0.9 μL of MNase. The cell lysate was incubated first for 20 minutes at 4 °C and then for 10 minutes at 37 °C. The MNase digestion was quenched by addition of 30 μL of 25 mM EGTA. Afterwards, the sample was immediately placed on ice and mixed ocassionally by inverting the tube for 2 minutes. The soluble chromatin was collected by spinning down the sample at 16000g for 5 minutes at 4 °C and mixed with the dilution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% DOC-Na, 2.2% Triton X-100, 50 mM EDTA, 50 mM EGTA, 5 mM Na-Butyrate). 2 % of the soluble fraction was taken as input and the rest was incubated overnight with the histone PTM antibody at 4 °C. The next day, magnetic Protein A or G Dynabeads were washed in PBS thrice and 20 μl per sample were added to collect the antibody-chromatin conjugates. Then, they were washed in the following buffers, each for 5 minutes at 4 °C: Low salt buffer (0.1 % SDS, 1 % Triton X-100, 0.1 % DOC-Na, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl)[twice], high salt buffer (0.1 % SDS, 1 % Triton X-100, 0.1 % DOC-Na, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 360 mM NaCl), LiCl buffer (0.25 M LiCl, 1 % NP- 40, 1 % DOC-Na. 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) [twice]. Finally, the samples were rinsed once in TE buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) and resuspended in 100 μL of elution buffer (50 mM Tris-HCl, pH 7.4, 1 % SDS, 1 mM EDTA) and incubated first for 1 h at 55 °C with Proteinase K and then for 30 minutes at 37 °C with RNAse A. Resulting DNA was purified using ChIP DNA Clean and Concentrator kit according to manufacturer's protocol.                                                                                                                                                        TT-Seq: TT-Seq was performed as previously described with minor modifications 28: ESCs were treated with 500 μM 4-sU for 5 minutes under regular growth conditions and lysed using Trizol (Invitrogen) immediately afterwards. Total RNA was purified using chloroform and isopropanol precipitation and resuspended in TE buffer. 300 μg of total RNA for each sample was spiked with spike-in mix (appendix) and fragmented using Bioruptor Plus (30 seconds ON, 30 seconds OFF, 1 cycle, HIGH setting) and an aliquot was taken as Total RNA sample. The rest of the fragmented total RNA was biotinylated using Biotin-HPDP (Thermo Fisher) at room temperature for 2 hours. To remove excess Biotin-HPDP, RNA was precipitated using phenol:chloroform-isoamyl alcohol (Sigma) and ethanol and resuspended in ddH2O. Biotinylated RNA fragments were purified using μMACS streptavidin beads and μMACS columns (Miltenyi Biotec) according to the recommendations of the manufacturer. Labeled RNA was eluted twice using 100 mM DTT and purified using Oligo Clean and Concentrator Kit (Zymo Research). Both total and labeled RNA samples were treated with Turbo DNAse (Invitrogen) for 30 minutes at 37 °C and purified again Oligo Clean and Concentrator Kit (Zymo Research). RNA libraries for total RNA and TT-Seq samples were prepared using Illumina Stranded Total RNA Prep Kit with RiboZero Plus. ChIP-Seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina.