GSM7813853: ES-Low-Rep3; Mus musculus; OTHER
Genomic DNA was extracted from the sorted cell populations using the Qiagen DNA Micro kit following manufacturer's instructions. To amplify the fragments contained in the reporter cassettes and add sequencing adaptors a PCR using indexed primers compatible with Illumina sequencing and targeted against the tn5 transposase sequence ((0.5 µM Nextera PCR Primer i5, 0.5 µM Nextera PCR Primer i7, 25 µl NEBNext Master Mix). Dependant on the amount of DNA obtained from the sorted cells between 25 and 30 PCR cycles were performed with the following conditions 98°C for 30 seconds, cycles of 98°C 10 seconds, 63°C 30 seconds and 72°C 1 minute with a final hold at 4°C. Libraries were quantified using a Bioanalyzer 2100 (Agilent) with a High Sensitivity DNA Chip and pooled at equal concentration for sequencing. The final pool concentration and size was then confirmed by Kappa Library Quantification Kit (Roche) and Bioanalyzer. The pooled libraries were sequenced on a Next-Seq 550 as a paired end run with a 150 cycle High Output kit by the Genomics Birmingham Sequencing Facility.