Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Cells were grown to 75% confluency in 150 mm culture dishes. Four hours prior to transfection, the medium was refreshed. To transfect, 30 μg of DNA (MmGtst1-TEV-Strep in pEF) was premixed with 60 μL of X-tremeGENE HP DNA transfection reagent (Roche) in serum-free medium for 15 minutes. After a 15-minute incubation, this mixture was added to the cultures. Forty-eight hours post-transfection, the cells were rinsed with ice-cold phosphate-buffered saline (PBS) and taken for crosslinking. UV cross-linking was performed at 254 nm for ~45 seconds (400 mJ) in an HL-2000 Hybrilinker. Crosslinked cells were harvested then stored frozen at -80°C until ready to proceed. Cell pellets were thawed in 1 mL of lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and protease inhibitors), resuspended by pipetting, then incubated on ice for 15 minutes. The cell suspension was further lysed using a Bioruptor ('low' setting at 4°C for 5 minutes [30 seconds on, 30 seconds off]). Two microliters of Turbo DNase (2 U/μL) (LifeTech) were added to each sample as well as 10 μL of RNase I (4 U/μL) (LifeTech) and mixed by pipetting. The samples were then incubated at 37°C for exactly 5 minutes with mixing (1200 rpm in a ThermoMixer). After the five-minute incubation, the samples were placed on ice and murine RNase inhibitor (40 U/μL) (NEB) was added immediately. Cell debris was removed by centrifugation at 15,000g for 15 minutes at 4°C and the clarified supernatants were transferred to fresh tubes. MagStrep type 3 XT beads (IBA) were equilibrated in 50 mM Tris, pH 8.0, 100 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate with protease inhibitors. Fifty microliters of bead resuspension were used per sample. Beads were added to each sample and incubated with gentle agitation at 4°C for 2.5 hours. Input samples were reserved for both sequencing and Western blotting; these samples were stored at 4°C until needed. Affinity-precipitated material was isolated by magnetic separation, followed by several wash steps. Libraries were generated essentially as described in Van Nostrand et al. (2016). Briefly, RNA ends were prepared by first treating with FastAP to remove terminal phosphates, then 5' phosphorylating using PNK. Barcoded adapters were then appended using 3' linker ligation with T4 ligase. Samples were then subjected to SDS-PAGE followed by blotting. Regions of the blot corresponding to the crosslinked protein of interest were cut out, and the RNA released from the membrane by Proteinase K treatment followed by nucleic acid extraction. Samples were reverse transcribed, RNA removed, and an additional barcoded adapter appended by 3' linker ligation. Samples were cleaned up using MyOne silane beads, then taken for final PCR amplification of the library. Libraries were pooled in equimolar ratios according to their quantification by Bioanalyzer. Paired-end reads with two, 8-basepair, barcodes were generated on an Illumina NextSeq resulting in approximately 100 million paired-end reads (~15-20 million reads per library).