title: GSM2261508: Input_SOX2_Mouse_Cortex; Mus musculus; ChIP-Seq
library_construction_protocol: Two-month old male wide-type mouse cortex tissue (0.3 g) was dissected on ice and cut into small pieces. Then the tissues were incubated 10 ml 1.5 mM ethylene glycolbis (succinimidyl succinate, EGTA) in a long-arm cross-linker buffer (15 mM Hepes-Cl 7.6, 60 mM KCl, 15 mM NaCl, 0.34 M sucrose, 2 mM MgCl2, 1 tab/10 mL EDTA free Protease inhibitor tablet) for 20 min at room temperature. The tissue was then crosslinked with at least ﬁve volumes of freshly prepared solution (1% formaldehyde, 50 mM Hepes-KOH, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 12 min. The reaction was quenched with 1/20 volume of 2.5 M glycine and the tissue was washed twice with ice-cold PBS. The tissue was dounced in ice-cold PBS ﬁrst with the loose and later with the tight pestle and then ﬁltered through a 100 um cell strainer to remove connective tissue. The tissue was collected by centrifuge at 4 ℃ at 2000 rcf for 5 min. Resuspend each pellet of cross linked tissue in 10 ml of lysis buffer 1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA;10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100, Proteinase inhibitor). Rock at 4 ℃ for 10 min. Spin at 2000 rcf for 4 min at 4 ℃. Resuspend each pellet in 10 ml of lysis buffer 2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA, Proteinase inhibitor). Rock gently at 4 ℃ for 5 min. Collected nuclei pellet by spinning at 2000 rcf for 5 min at 4 ℃. Resuspended nuclei pellet in shearing buffer (50 mM HEPES [pH 8.0], 10 mM EDTA [pH 8.0], 1% SDS, EDTA-free complete protease inhibitor), and chromatin sheared for 45 min of 30 s ON and 30 s OFF until DNA fragments were 200-600 bp. Sheared chromatin (50 mg) was diluted 10-fold in immunoprecipitation buffer (50 mM HEPES (pH 8.0], 20 mM NaCl, 1 mM EDTA [pH 8.0], 0.1% Triton X-100, EDTA-free complete protease inhibitor) and immunoprecipitated with anti-SOX2 (AB5603, rabbit, 5 mg, Millipore) at 4℃ for 14 hr. Protein G magnetic Dynabeads (100 ml, Life Technologies) were used to isolate immunoprecipitated chromatin at 4℃ for 2 hr. The immunoprecipitated fraction was washed twice with immunoprecipitation buffer, twice with wash buffer (100 mM Tris-HCl [pH 9.0], 500 mM LiCl, 1% IGEPAL CA-630, 1% deoxycholic acid, and EDTA-free complete protease inhibitor) and one time with high salt wash buffer (wash buffer containing 150 mM NaCl). Chromatin was eluted in 100 ml elution buffer (1% SDS, 50 mM sodium bicarbonate) at 25℃ for 30 min with shaking. Eluted chromatin was treated with 1 ml RNase A (10 mg/ml) and 2 ml Proteinase K (40 mg/ml) at 37℃ for 1 hr, and then then crosslinking was reversed with NaCl (333 mM ﬁnal concentration) at 67℃ for 14 hr. Immunoprecipitated DNA was puriﬁed using the QIAquick PCR Puriﬁcation Kit (QIAGEN) according to the manufacturer’s speciﬁcations. Quantitative real-time PCR was performed to determine the foldenrichment of SOX2 immunoprecitated DNA relative to background in Input samples. ChIP experiments were repeated for three times, and three mice were used for each Immunoprecipitated. Libraries were synthesized from 5 ng purified input chromatin and 5 ng purified immunoprecipitated chromatin using a NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, E6240S) with NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S). Replicate libraries were prepared from independent immunoprecipitations. ChIP-seq: single end 50-base length sequencing reads were generated on an Illumina HiSeq 2500 System.
attributes.tag: GEO Accession
attributes.value: GSM2261508