GSM5768758: RNAseq_siC_G1_day9_EBs_R2; Mus musculus; RNA-Seq
RNA-seq: RNA extraction was performed using the RNeasy mini kit (Qiagen) following manufacturer's instruction. RNA samples were quantified and quality was evaluated using Bioanalyzer (RIN > 9.9). A total of 0.5 ug of RNA was used as starting material for library preparation with rRNA depletion using TruSeq Stranded Total RNA Library Prep kit (Ilumina) following the manufacturer´s instruction. modified DNA immunoprecipitation followed by sequencing (DIP-seq): DNA extraction and purification was carried out as in (PMID: 30220521). Briefly, DNA was extracted using the Blood & Cell Culture DNA Mini kit (QIAGEN Cat#13323). Purified genomic DNA was sonicated using Covaris to obtain fragments of 200–600 bp. Library preparation was performed using the NEBNext Ultra library prep kit (New England Biolabs Cat#E7370) substituting the NEB hairpin adaptors by full-length barcoded TruSeq adaptors from Illumina (Cat# FC-401-3001). Fragmented DNA with compatible adaptors were pooled together. Pooled DNA was heated for 10 minutes at 99°C, cold down on ice, and incubated overnight with 5-Carboxylcytosine (5-caC) antibody (1 μg Ab/μg DNA; Active Motif Cat#61225) in IP buffer (10 mM Sodium Phosphate buffer, 140 mM NaCl, 0.05% Triton X-100). Immunocomplexes were recovered using pre-equilibrated Protein G/A Dynabeads (Life Technologies Cat#10002D and Cat#10009D respectively) for 2 hr at 4°C. After washing with IP buffer, immunocomplexes were eluted from columns by heating at 55°C for 30 minutes in proteinase digestion buffer [50 mM Tris-HCl pH = 8, 10 mM EDTA, 0.5% SDS, 35 mg Proteinase K (New England Biolabs Cat#P8107S)] and the DNA purified using MinElute PCR Purification kit (QIAGEN Cat#28004). Recovered DNA was amplified by PCR and sequenced in Illumina HiSeq2500. Libraries were prepared according to Illumina instructions.