GSM6447783: RPE-1 hTERT FUCCI single pulse 15min_6; Homo sapiens; OTHER
TrypLE digestion Custom library protocol. Briefly, EdU was clicked to Biotin-PEG3-Azide. Single cells were sorted in a 384-well plate. Subsequently, cells were lysed wioth ProtK and the genome was digested with NLAIII. Next, DNA fgragments were end-repaired by Klenow Large Fragment and PNK. On these repaired ends we ligated a custom adapter sequence followed by affininty purification of EdU-Biotin miolecules with magnetic streptadin beads. Non-modified strands were recovered by heat denaturation and the complimetary strand was re-generated by a primer extension reaction. Finally, libraries were amplified by a combination of T7 in vitro trasncription, reverse transcription and polymerase chain reaction containing Illumina handles Illumina RPI small RNA adapters