Cells are crosslinked with 0.15mJ/cm2 UV-C irradiation, lysed with 1xNLB and homogenized by waterbath-sonication. Target protein of interest is pulled-down with paramagnetic beads pre-coupled to antibodies against the protein of interest. After a brief RNAseI-digestion, RNA 3'-ends are healed with T4 PNK. Custom-made, barcoded adapters are ligated using T4 RNA Ligase 1 or T4 RNA Ligase 2KQ (if pre-adenylated adapters are used) 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 are used to merge PCR-duplicates, regular tags are used to specify the pulldown condition, and semi-random RY-space tags are used to distinguish the biological replicates (RR: replicate A, YY: replicate B, R: purine, Y: pyrimidine). Excess adapters are washed away, negative controls (IgG) are mixed with experimental controls and RNA is isolated with Proteinase K treatment and column purification. Isolated RNA is reverse-transcribed and RNaseH-treated. cDNA is column-purified and circularized with CircLigase for 2-16hrs. Circularized cDNA is directly PCR amplified, quantified with Qubit / Bioanalyzer and sequenced on Illumina NextSeq 500 in paired-end mode.
GEO Accession
GSM2363522