title: GSM1612345: Tet2 -/- proliferating Neural Stem Cell.5hmC-1; Mus musculus; MeDIP-Seq
library_construction_protocol: Five to ten million NSCs were treated with 1% formaldehyde for 10 min at room temperature with gentle shaking. Fixation was terminated by adding 2M Glycine to reach 0.125M final concentration, and continue shaking for additional 5min. Cells were collected by cell scrapers and spin down at 1500 rpm for 10 min.  Cell pellet was resuspended in 1ml Nuclei Swelling Buffer (10 mM Hepes/pH7.9, 0.5% NP-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and protease inhibitor cocktail), incubated on ice for 10 min and centrifuged at 5000 rpm for 5 min. Nuclear pellets were further lysed in 1ml SDS lysis buffer (20 mM Hepes/pH7.9, 25% glycerol, 0.5% NP-40, 0.5% Triton X-100, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and protease inhibitor cocktail). Cell lysate were sonicated 5 times with 6-9W output to obtain DNA fragments between 200-500bp. After sonication, nuclear lysate was cleared by centrifugation at 13000 rpm for 10 min to keep supernatant. The nuclear lysate was diluted with 3-4 volumes of dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl/pH8.0 and protease inhibitor cocktail). Immunoprecipitation was performed with antibodies specific to Foxo3a (1:1000, Bethyl) rotating overnight at 4ºC. 20 μl Protein-G Dynabeads (Life technologies) were added for additional 2 hours. TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl/pH 8.1), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl/pH 8.1), TSE III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl/pH 8.1), and washed twice with TE buffer before eluted with 1% SDS and 0.1 M NaHCO3. The elution was incubated at 65 ºC for at least 6 hours to reverse the formaldehyde cross-linking, then DNA fragments were purified by using PCR Purification Kit (Qiagen). ChIPed DNA was subjected to library preparation. For genomic DNA extraction, Genomic DNA was first sonicated to 100-500bp, and mixed with 100 ml solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 250 mM UDP-6-N3-Glu and 2.25 mM wild-type b-glucosyltransferase. Reactions were incubated for 1 h at 37°C. DNA substrates were purified via Qiagen DNA purification kit. 150 mM dibenzocyclooctyne modified biotin was then added to the purified DNA, and the labeling re- action was performed for 2 h at 37°C. The biotin-labeled DNA was enriched by Streptavidin-coupled Dynabeads (Dynabeadsw MyOneTM Streptavidin T1, Life Technologies) and purified by Qiagen DNA purification kit for library preparation. RNA isolation was done by Trizol. 25ng of input genomic DNA or experimental enriched DNA were utilized for each library construction. 150-300 bp DNA fragments were selected by AMPure XP Beads (Beckman Coulter) after the adapter ligation. An Agilent 2100 BioAnalyzer was used to quantify the amplified DNA, qPCR was applied to accurately quantify the library concentration. 20 pM diluted libraries were used for sequencing. 50-cycle single-end sequencings were performed using Illumina HISeq 2000. Image processing and sequence extraction were done using the standard Illumina Pipeline. RNA-seq libraries were generated from duplicated samples per condition using the Illumina TruSeq RNA Sample Preparation Kit v2 following manufacturer’s protocol. The RNA-seq libraries were sequenced as 50-cycle pair-end runs using Illumina HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM1612345