GSM6784541: Hippocampus, WT, size‐matched input, rep2; Mus musculus; RIP-Seq
The CLIP experiments were performed according to the eCLIP protocol from Nostrand et al. (Van Nostrand et al., 2016) with some modifications. After IP, beads were washed 2x with the high salt wash buffer (50mM Tris-HCl pH7.5, 1M NaCl, 1mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), 2x CLIP-lysis buffer, 2x with low salt wash buffer (20mM Tris-HCl pH7.5, 10mM MgCl2, 0.2% Tween-20) and 1x with PNK buffer 70mM Tris-HCl pH6.5, 10mM MgCl2). Beads were re-suspended in 100µl PNK-mix (70mM Tris-HCl pH6.5, 10mM MgCl2, 1mM DTT, 100U RNasin, 1U TurboDNase, 25U Polynucleotide-Kinase (NEB)) and incubated for 20min at 37°C in a thermomixer with shaking (1200 x rpm). After RNA dephosphorylation beads were washed as before with 2x high salt, 2x lysis and 2x low salt buffers and additionally with 1x Ligase buffer (50mM Tris-HCl pH7.5, 10mM MgCl2). Beads were re-suspended in 50µl ligase mix (50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM ATP, 3 % DMSO, 15% PEG8000, 30U RNasin, 75U T4 RNA-ligase (NEB)). 10µl of the beads / ligase mix were transferred to a new tube and 1µl of pCp-Biotin (Jena Bioscience) were added to validate IP of the RNA-protein-complexes by western blot. To the rest (40µl) 4µl of the RNA-adaptor mix containing 40µM of each RNA_X1A & RNA_X1B (IDT) were added and samples were incubated for 2h at RT. After adaptor ligation samples were washed 2x with high salt, 2x with lysis and 1x with low salt buffers. Beads were re-suspended in 1x LDS sample buffer (Thermofisher) supplemented with 10 uM DTT and incubated at 65°C for 10min with shaking at 1200 x rpm. Eluates or inputs were loaded on 4-12% Bis-Tris, 10-well, 1.5mm gel (Thermofisher) and separated at 130V for ca. 1.5h. Proteins were transferred to the nitrocellulose membrane (Amersham) overnight at 30V. After transfer the membranes were placed in a 15cm Petri dish on ice and an area between 55 and 145kDa was cut out in small pieces and transferred to 2ml tube. For CLIP samples RNA extraction, reverse transcription using AR17 primer, cDNA clean-up using silane beads (Thermofisher), second adaptor ligation (rand103Tr3) and final cDNA purification were performed as previously described (Van Nostrand et al., 2016). For sized matched input samples (SMIn) isolated RNA was dephosphorylated. The sequencing libraries were amplified using Q5-DNA polymerase (NEB) and i50X/i70X Illumina indexing primers (IDT). The amplified libraries were purified and concentrated first with ProNEX size selective purification system (Promega) using sample/beads ratio of 1/2.4. The purified libraries were loaded on a 2% agarose gel, the area corresponding to the size between 175bp and 350bp was cut and the libraries were extracted from the gel using gel extraction kit (Machery&Nagel) and eluted with 16µl. Concentrations and size distributions of the libraries were determined on Fragment analyzer system (Agilent). 75bp paired-end sequencing was performed on the NextSeq500 platform using Mid Output Kit v2.5 (150 cycles). Eric L. Van Nostrand et al. 2016 eCLIP-Seq