GSM6637978: 25-32-exp1; Homo sapiens; OTHER
The harvested cells were lysed in 1 ml of TRI Reagent™ Solution (Ambion) and total RNA was isolated according to manufacturer's instructions. Ten μg of purified RNA was incubated with 3 U of DNase I (Thermo Fisher Scientific) and 40 U of DpnI restriction enzyme (New England Biolabs) at 37 °C for 30 min to remove traces of genomic and plasmid DNA. The RNA was purified using CleanRNA Standard kit (Evrogen) according to the manufacturer's instructions. Two μg of purified total RNA were reverse-transcribed into cDNA using anchored oligo(dT)20 primer and 100 U of RevertAid Reverse Transcriptase (Thermo Fisher Scientific) according to manufacturer's instructions. The mapping samples were prepared according to a two-round conventional PCR using primers specific to the ends of fixed sequences mCP1 and mCP3. The expression samples were prepared by PCR amplification of the BC sequences using 1/20 of cDNA as a template and primers specific to the ends of fixed sequences neCP1 and neCP2. The normalization samples were prepared by PCR amplification of the using plasmid DNA as a template and primers specific to the ends of fixed sequences neCP1 and neCP2.