GSM6260051: iPSC-neurons, PTBP2 eCLIP, rep3; Homo sapiens; RIP-Seq
Crosslinked neurons or brain tissues were lysed in eCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate) containing proteinase inhibitor and RNAse inhibitor. Cell or tissue lysis was assisted with sonication by using a Bioruptor on the low setting for 5 min, cycling 30 s on and 30 s off.  For each 1 mL lysate, 5 µL Turbo DNAse I (Thermo Fisher #AM2239) and high dose RNAse I (Thermo Fisher #AM2295; 1:5 diluted in cold DPBS) or low dose RNAse I (1:50 dilution for neurons, and 1:100 dilution for brain) were added. The microspin tubes were placed in a Thermomixer preheated to 37 °C for exactly 5 min, shaken at 1,200 rpm, and then put on ice to terminate the reaction. Immunoprecipitated-RNA was dephosphorylated with FastAP enzyme (Thermo Scientific #EF0652) and T4 PNK (New England BioLabs #M0201L), and an adapter labeled with an IRDye®800CW fluorochrome (/5Phos/rNrNrNrN rArGrA rUrCrG rGrArA rGrArG rCrArC rArCrG rUrCrU rGrArA rArA/3IR800CWN/) was ligated to the 3′ end. Labeled RBP-RNA complexes were eluted using 1x LDS loading buffer (Thermo Fisher) and resolved on 4-12% Bis-Tris gels, transferred to nitrocellulose membrane, and imaged. RNA-protein complexes were visualized with LI-COR Odyssey imaging system, and a region including ~75 kDa above protein size (by comparing with high RNAse I sample) was cut from the membrane. RNA was isolated from the membrane via protease K/SDS treatment. After reverse transcription with a modified primer (5'-TTC AGA CGT GTG CTC TTC CG-3'), a 5′ adapter containing 8-nt UMI (/5phos/NNNNNNNN AGATCGGAAGAGCGTCGTGTAGGG/3ddC/) was ligated to cDNA. All of the remaining steps were essentially performed according to the eCLIP procedure (van Nostrand et al., 2016).