GSM6510272: EpiChromo_Screen_maintenance_eGFP+_rep1; Mus musculus; OTHER
Cell pellets from eGFP sorted and unsorted cells were treated with proteinase K and DNAse-free RNase A in lysis buffer (50 mM KCl, 10 mM Tris-HCl (pH8.3), 2.5 mM MgCl2-6 H2O, 0.45% NP-40, 0.45% Tween-20) and genomic DNA (gDNA) was initially purified using phenol/chloroform/isoamyl alcohol and then ethanol precipitated. PCR of gDNA was performed to attach sequencing adaptors and barcode samples. gDNA of each sample was divided into multiple 50 μl reactions containing a maximum of 2.5 μg gDNA per reaction. For each PCR reaction we used: 0.75 μl of ExTaq DNA polymerase (Clontech), 5 μl of (10x) ExTaq buffer, 4 μl of dNTPs provided with the enzyme, 2.5 μl of P5 stagger primer mix (stock at 10 μM) and 2.5 μl of P7 uniquely barcoded primer (stock at 10 μM). PCR cycling conditions were an initial 3 min at 95 °C; followed by 30 s at 95 °C, 30 s at 53 °C, 30 s at 72 °C, for 23 cycles; and a final 10 min extension at 72 °C. PCR products for each sample were pooled, electrophoresed in an agarose gel, purified (NucleoSpin® Extract II) and sequenced on an Illumina NextSeq 500