title: GSM3212766: 5hmC Tet2-KO; Mus musculus; MeDIP-Seq
library_construction_protocol: Total RNA was isolated from cell pellets by Trizol reagent and genomic DNA was prepared using a Genomic DNA Purification Kit (Promega) RNA-Seq library were generated using KAPA Strandard mRNA-Seq Kits according to the manufacturer's recommendations (Cat No. KK8440 box1 and KK8420 box2). Briefly, the mRNA was enriched using oligo(dT) magnetic beads and sheared to create short fragments of approximately 300 bp. cDNA was synthesized using random hexamer primers and purified using 1.8X Agencourt AMPure XP beads (Beckman). Finally, the sequencing primers linked to the cDNA fragments (approximately 300 bp) were isolated by gel electrophoresis and enriched by PCR amplification to construct the library. The sequencing was performed at the Sequencing Center in National Institute of Biology, Beijing using the HiSeq 2500 system developed by Illumina. Paired-end 100-bp sequencing was applied. For (h)MeDIP-seq, 3 μg of genomic DNA was used to prepare the sample for sonicated fragmentation. After sonication of genomic DNA the ends of the DNA fragments were repaired and paired-end sequencing specific adaptors (Illumina) were ligated using either a Paired-End DNA Sample Preparation Kit (Illumina) or NEBNext DNA Sample Prep Reagent Set 1 (NEB). Following adaptor ligation, 1/50 of each sample was saved as input, the other DNA was immunoprecipitated and purified. Fragments were amplified with 12–18 cycles using adaptor specific primers (Illumina); fragments ranging between 200 and 500 bp in size were gel-purified before cluster generation and sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3212766