GSM5942201: Expt5_293T_input_total_rep1; Homo sapiens; OTHER
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 seCLIP protocol (Van Nostrand et al., 2016 & 2017) with modifications to enhance chimera formation described below. As in eCLIP, lysis was performed in eCLIP lysis buffer, followed by sonication and digestion with RNase I (Ambion). Immunoprecipitation of AGO2-RNA complexes was achieved with a primary mouse monoclonal Ago2 antibody (eIF2C2 (4F9) Santa Cruz, 4°C overnight) using magnetic beads pre-coupled to the secondary antibody (M-280 Sheep Anti-Mouse IgG Dynabeads, Thermo Fisher 11202D). Initial experiments used standard eCLIP conditions (10 ug of antibody and 125 uL of Dynabeads for 20×10^6 cells), but most experiments used decreased antibody and increased bead amounts based on the trend of decreased cross-species chimeras in those conditions (See Sup. Fig. 2X and Sup. Table X). Where indicated, 2% of each immunoprecipitated (IP) sample was saved as input control. For human/rat mixing experiments, cell pellets were lysed, sonicated, and RNase digested separately, and then mixed during addition of antibody and beads prior to overnight incubation. To phosphorylate the cleaved mRNA 5'-ends, beads were washed and treated with T4 polynucleotide kinase (PNK, 3' -phosphatase minus, NEB) and 1 mM ATP. Chimeric ligation was then performed on-bead at room temperature for one hour with T4 RNA Ligase I (NEB) and 1 mM ATP in a 150 µl total volume. As in seCLIP, samples were then dephosphorylated with alkaline phosphatase (FastAP, Thermo Fisher) and T4 PNK (NEB), and an RNA adapter was ligated to the 3′-ends of the mRNA fragments (T4 RNA Ligase, NEB). With-gel chimeric-eCLIP IP and input 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. The region corresponding to bands at the appropriate Ago2 protein size plus 75 kDa was excised and treated with Proteinase K (NEB) to isolate RNA, which was column purified (Zymo). No-gel chimeric eCLIP samples were treated directly with Proteinase K (NEB) to isolate RNA and column purified (Zymo). For both methods, RNA was then reverse transcribed with SuperScript IV Reverse Transcriptase (Invitrogen), 3 mM manganese chloride (to encourage read-through of crosslink sites), and 0.1 M DTT. Following reverse transcription, samples were treated as in seCLIP, including treatment with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides, hydrolysis with sodium hydroxide (to degrade RNA) and addition of hydrogen chloride (to balance pH). A 5' Illumina DNA adapter (/5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3) was then ligated to the 3′-end of cDNA fragments with T4 RNA Ligase (NEB), and after bead purification (Dynabeads MyOne Silane, Thermo Fisher), qPCR was performed on an aliquot of each sample to identify the proper number of PCR cycles. The remainder of the sample was PCR amplified with barcoded Illumina compatible primers (Q5, NEB) based on qPCR quantification and size selected using AMPure XP beads (Beckman). Libraries were quantified using Agilent TapeStation and sequenced on the Illumina HiSeq or NovaSeq platform.