GSM5742139: FLASH GFP rep1; Homo sapiens; OTHER
FLASH-seq protocol was applied as described in Aktaş et al., 2017. Briefly, cells were crosslinked with 0.2mJ/cm2 UV-C irradiation, lysed with 1xNLB and homogenized by waterbath-sonication. Target protein of interest were affinity purified in tandem with nickel-charged paramagnetic beads and streptavidin-coupled paramagnetic beads against the tagged protein of interest. After a brief RNAseI-digestion, RNA 3'-ends were healed with T4 PNK. Custom-made, barcoded adapters were ligated using T4 RNA Ligase 1, for 1 hr at 25˚C. Custom FLASH adapters contained two barcodes and random nucleotides adjacent to the 3'-adapters according to the pattern NNBBNTTTTTTNN (N: random tag nucleotide, T: tag nucleotide, B: RY-space tag nucleotide). Random tags were used to merge PCR-duplicates, regular tags were used to specify the pulldown condition, and semi-random RY-space tags were used to distinguish the biological replicates (RR: replicate A, YY: replicate B, R: purine, Y: pyrimidine). Excess adapters were washed away and RNA was isolated with Proteinase K treatment and column purification. Isolated RNA was reverse-transcribed and RNaseH-treated. cDNA was column-purified and circularized with CircLigase for 16hrs. Circularized cDNA was directly PCR amplified, quantified with Qubit / Bioanalyzer and sequenced on Illumina NextSeq 500 in paired-end mode. Libraries were prepared according to NEBNext Ultra II DNA Library preparation kit (NEB #E7645S)