title: GSM8252115: Hela_TRMT1L_IP_Replicate1; Homo sapiens; OTHER
library_construction_protocol: The eCLIP-sequencing pipeline was adapted from procedures described by Van Nostrand et al. (Methods Mol. Biol., 2017, 1648, 177-200). Cells were initially crosslinked with 400 mJ/cm2 UV on ice and lysed in CLIP buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1× protease inhibitor cocktail) for 15 min. RNA fragmentation was accomplished through sonication, followed by treatment with RNase T1 and Turbo DNase at 37°C for 5 minutes. The nuclease-treated lysate was immunoprecipitated with protein G Dynabeads pre-bound with anti-TRMT1L antibody or rabbit IgG overnight. The co-precipitated complex was washed sequentially with high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1% NP-40,0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA) and wash buffer (20 mM Tris-HCl pH 7.4, 0.2% Tween-20). The RNA-ends were repaired on beads by treatment with Fast-AP and T4 PNK. The eCLIP-sequencing pipeline was adapted from procedures described by Van Nostrand et al. (Methods Mol. Biol., 2017, 1648, 177-200). The dephosphorylated RNA was ligated to a 3' RNA adaptor (/5Phos/rArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrC/3SpC3/) by T4 RNA ligase on beads. The co-precipitated complex and the samples for SMInputs were resolved on an 8% SDS-PAGE gel and transferred to a nitrocellulose membrane for size selection. Samples ranging from the TRMT1L band to 75 kDa above on the membrane were excised, recovered, and prepared into libraries for paired-end sequencing on an Illumina Hi-Seq 4000 platform (Novogene).