GSM2070058: CG3800 CLIPseq replicate 1; Drosophila melanogaster; OTHER
CLIPseq was performed in biological replicates and was essentially described in (Hafner et al. 2010). Buffers and RNA digest were adapted from (Huppertz et al. 2014). The cell lysate was thawed on ice and RNAseI (333U/ml lysate; 1:333 (v/v)) and TurboDNase (4U/ml lysate) treated for 3.5min at 37°C and 1100rmp and immediately replaced on ice for >3min. Lysates were cleared by centrifugation at 20,000g at 4°C. Immunoprecipitation (IP) was carried out with monoclonal FlagM2 (SIGMA) coated magnetic proteinG dynabeads (Life) on spin-cleared cell extracts for 2 h at 4ºC. Before IP, 50µl of the cleared lysate was taken, proteinase K treated and used for RNA extraction (Zymo DirectZol RNA-Mini Prep Kit). 2.5µg of this fragmented input total RNA was Ribozero treated. 100ng of rRNA-depleted, radioactively end-labeled input RNA was used to construct CLIP input libraries and resumed to CLIP cloning procedure at the step of 3’adapter ligation and was therefore subjected to the same RNA fragment size selection and adapter ligation procedures (see below).  After IP, CLIP samples were stringently washed 3 times with high salt buffer (1M NaCl). Samples were radioactively end-labeled with γ-32P-ATP (25min at 37°C and 1100rpm) and subsequent addition of 1µl high molar ATP for efficient 5’end phosphorylation. The crosslinked protein-RNA complexes were resolved on a 4-12% NuPAGE gel (Invitrogen). The SDS-PAGE gel was transferred to a nitrocellulose membrane (BioRad) and the protein-RNA complex migrating at an expected molecular weight was excised. RNA was isolated by Proteinase K (Roche) treatment and phenol-chloroform extraction, ligated to 3′ adapter (NNNN-TGGAATTCTCGGGTGCCAAGG) and 5′ adapter (GTTCAGAGTTCTACAGTCCGACGATC-NNNN), reverse transcribed, PCR-amplified (PCR cycles for libraries: PEP_CLIP_R1 und R2 18 x cycles; PEP_INPUT_R1 und R2 10 x cycles; CG3800_CLIP_R1 und R2 14 x cycles, CG3800_INPUT_R1 und R2 10 x cycles), and gel-excised. The amplified PEP cDNA was sequenced on HighSeq2000 (Illumina) with a 1x51 nt cycle. CG3800 cDNA was sequenced on NextSeq500 (Illumina) with 75 cycles single-end high output. Each CLIP was conducted in a biological replicate including a sized-matched input RNA library.