GSM4455997: eeTAPS 1ng; Mus musculus; OTHER
Confluent E14 mESC were dissociated from the plates and collected by centrifugation for 5 min at 1000 xg. Genomic DNA was extracted from 40 mln cells with Quick-DNA Plus kit (Zymo Research) according to manufacturer's protocol. To prepare eeTAPS libraries, DNA was converted with TAPS protocol, digested by USER enzyme and size selected to retain fragments 200-1000 bp. Final sequencing libraries were prepared with KAPA HyperPlus kit according to the manufacturer's protocol. The final sequencing libraries were amplified with KAPA HiFi HotStart ReadyMix for 6 cycles (200 ng input), 8 cycles (50 ng input), 10 cycles (10 ng input) or 14 cycles (1 ng input). For control library untreated DNA was digested by USER enzyme and used for library construction as described above. Final library was amplified for 6 cycles. To prepare rrTAPS library DNA was first digested by MspI enzyme then used for End-repair and A-tailing reaction and ligation of Illumina Multiplexing adapters were prepared with NEBNext® Ultra™ II DNA Library Prep Kit according to the manufacturer's protocol. DNA fragments from 100-400 bp were cut out of the gel and purified by Monarch® DNA Gel Extraction Kit following manufacturer's protocol. Adapter-ligated sample was TAPS-converted and amplified with KAPA HiFi Uracil (+) Master Mix for 6 cycles.