GSM2973501: Dmel_female_MLE; Drosophila melanogaster; RIP-Seq
Larvae were dissected and the front part was homogenized and UV crossllinked at 0.15 mJ/cm2. The homogenate was cleared using a sucrose cushion followed by 10 cycles of sonication in a Bioruptor Pico. The lysate was clarified by centrifugation before pull-down of the protein of interest. After a brief RNaseI-digestion, RNA 3'-ends were healed with T4-PNK. Custom-made barcoded adapters are ligated using T4 RNA Ligase1 for 1 hr at 25˚C. Custom FLASH adapters contained index barcodes and random nucleotides adjacent to the 3'-adapters according to the pattern NNNNNTTTTTTNN (N: random tag nucleotide, T: index nucleotide). Index nucleotides were used to separate samples. Random tags were used to merge PCR-duplicates. Excess adapters are washed away 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 or HiSeq 3000 in paired-end mode.