GSM6482955: DamID_Damonly_ESC_G4_E35_mm10_dRex1_dTrim; Mus musculus; OTHER
DNA  was  extracted  from  cells  using  the  DNeasy  tissue  lysis  kit  as  per  manufacturer's  instructions. DamID  sample  processing  was  then  performed  as  described previously (Robson and Schirmer, 2016).  Briefly,   2.5  μg  of  extracted  DNA  was  then  digested by DpnI and, following heat inactivation of DpnI,  was ligated to the DamID adaptor duplex (dsAdR) generated from the oligonucleotides AdRt (5'-CTAATACGACTCACATAGGGCAGCGTGGTCGCGGCCGA-GGA-3')  and AdRb (5'-TCCTCGGCCG-3') after which DNA was further digested by DpnII. To amplify DNA sequences methylated by the Dam methylase, 5 μl of DpnII digested material was then subjected to PCR in the supplied buffer in the presence of the 1.25 μM Adr-PCR primer (5'-GGTCGCGGCCGAGGATC-3'), 0.2 mM dNTPs and 1X of the Advantage cDNA polymerase. PCR was performed as previously described after which amplified DNA was purified, processed into NGS libraries using the KAPA HyperPrep kit and analyzed for quality by Bioanalyzer analysis.  standard protocols. DamID-seq samples were sequenced 75 or 100 bp paired-end reads and each experiment was performed in duplicates for sequencing.