title: GSM8521928: NOLC1 chimeric eCLIP Input in HepG2; Homo sapiens; OTHER
library_construction_protocol: For each 10cm plate (~15 million cells), cells were washed once with cold phosphate buffered saline, and then UV crosslinked (254 nm, 400 mJ/cm2) on ice. Cells were then spun down, supernatant removed, and washed with cold phosphate buffered saline. Cell pellets were flash frozen on dry ice and stored at -80°C. Chimeric eCLIP was based off the previously described chimeric eCLIP protocol (Manakov et al. https://doi.org/10.1101/2022.02.13.480296). Lysis was performed in eCLIP lysis buffer, followed by sonication and digestion with RNase I (Ambion). Immunoprecipitation of snoRNA-associated RBPs-RNA complexes was achieved with a primary antibody using magnetic beads pre-coupled to the secondary antibody (typically 200 ul M-280 Sheep Anti-Rabbit IgG Dynabeads, Thermo Fisher Scientific). Where indicated, 2% of each immunoprecipitated (IP) sample was saved as input control. RNA fragments was dephosphorylated with FastAP (Thermo Fisher) and T4 PNK (NEB), followed by 5' end phosphorylation of cleaved target RNAs with T4 polynucleotide kinase (PNK, 3' -phosphatase minus, NEB)). Chimeric ligation was then performed with T4 RNA ligase (NEB) without adapters. After additional dephosphorylation steps (FastAP, T4 PNK), phosphorylated RNA adapter was ligated to the 3' end (T4 RNA Ligase, NEB). Samples were then denatured with 1X NuPage buffer (Life Technologies) and DTT, run on 4%–12% Bis-Tris protein gels and transferred to nitrocellulose membranes(GE Amersham). The region from the observed protein size to 75kD above (corresponding to ~250 nt RNA) was then isolated from membrane by razor blade, and RNA was isolated by Proteinase K (NEB) treatment followed by column purification (Zymo). RNA was then reverse transcribed with SuperScript IV Reverse Transcriptase (Invitrogen) using manganese chloride-based buffer to enhance read-through of modified nucleotides [[ref]]. Excess primers were removed with ExoSAP-IT (Thermo Fisher), and RNA was degraded by alkaline hydrolysis, followed by RNA purification (MyOne Silane beads, Thermo Fisher). Next, a 5' Illumina DNA adapter (/5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3)  was ligated to the 3' end of cDNA fragment with T4 RNA ligase HC enzyme on beads overnight. After beads cleanup, an aliquot of each sample was proceeded for qPCR to identify the proper PCR cycles. The remainder of the samples were amplified with barcoded Illumina primers (Q5, NEB) and size selected via AMPure XP beads and 2% agarose gel (195-350bp). Samples were then quantified by Agilent TapeStation and sequenced either on the Illumina NextSeq (150 bp SE) or NovaSeq X Plus (150 bp PE) platforms.