GSM1159715: EpiSC_3_FT_9.73_p15_RNA_Seq; Mus musculus; RNA-Seq
Genomic DNA was purified using DNA extraction kit (Promega) according to the manufacturer’s instructions. DNA was extracted pools of seven E6.5 epiblasts or pools of three E7 epiblasts. After proteinase K digestion in lysis saline buffer DNA was extracted using NaCl/EtOH precipitation.Genomic DNA was sonicated to generate 300 bp fragments on average. MethylCap was performed using the IP-STAR robot (Diagenode) as described before (Bock et al., 2010).  In short, 1 μg DNA was incubated with paramagnetic beads coated with the MBD domain of MeCP2 fused to GST. After washing with 200 mM, 400 mM and 500 mM NaCl, the bound methylated DNA was eluted in two fractions using 600 mM and 800 mM NaCl, respectively. 20 ng of DNA eluates was prepared for sequencing. For RNA-Seq, Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s recommendations. 100 μg total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN). 100–200 ng mRNA was fragmented by hydrolysis (5x fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s and purified (RNAeasy Minelute Kit; QIAGEN). cDNA was synthesized using 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). Double-strand cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer’s recommendations and purified (Minelute Reaction Cleanup Kit; QIAGEN). 20 ng was used for sequencing. DNA or cDNA samples were prepared for sequencing by end repair of 20 ng DNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Integrity of DNA libraries was confirmed by running the products on a Bioanalyzer (BioRad). Cluster generation and sequencing (36 bp) was performed with the Illumina Genome Analyzer IIx (GAIIx) (MethylCap-seq) or HiSeq (RNA-seq) platform according to standard Illumina protocols.
GEO Accession
GSM1159715