title: GSM1915721: E14_Lin28KD_5mC_seq.rep1; Mus musculus; MBD-Seq
library_construction_protocol: Total RNA was extracted from mESCs by RNeasy (Qiagen), and 1 ug total RNA was primed with Oligo(dT)20 primers (Invitrogen) for cDNA synthesis. For 5hmC capture and sequencing, five ug of genomic DNA was sonicated to 100-500 bp, and then mixed with 100 ul reaction buffer (50 mM HEPES at pH 8.0, 25 mM MgCl2, 250 uM UDP-6-N3-Glu and 2.25 uM wild-type beta-glucosyltransferase (beta-GT)). Reactions were incubated at 37°C for 1 hour, and DNA substrates were then purified by Qiagen DNA purification kit. 150 uM dibenzocyclooctyne modified biotin was mixed with beta-GT-modified DNA. The labelling reaction was performed at 37°C for 2 hours. The biotin-labeled DNA was enriched by Strepavidin-coupled Dynabeads (Dynabeads MyOneTM Streptavidin T1, Life Technologies) and purified by Qiagen DNA purification kit. 5mC experiments were performed according to the manufacturer's protocol (Active Motif). Briefly, five microgram of DNA was sonicated to 100-500bp, and then mixed with 10ul buffer C and water to form 95ul total pulldown reaction. 2ul 5mC antibody was incubated with sonicated DNA overnight. 25ul Protein G magnetic beads were added to capture 5mC antibody and its bound mC containing DNA. Wash 3x with buffer C and puriy the DNA with Qiagen PCR purification kit. The enriched methylated DNA was purified by Qiagen DNA purification kit. 5hmC- and 5mC-containing DNA was subjected to library preparation. 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: GSM1915721