GSM9092797: V. fischeri, VF_2432-3XFLAG, Replicate 2 (iCLIP); Aliivibrio fischeri; OTHER
Both crosslinked aliquots were decanted into a 50 mL conical and centrifuged for 15 min at 6,000 x g and 4 ˚C. Supernatants were discarded and pellets were resuspended in 4 mL ice cold Lysis Buffer [50 mM Tris-HCl pH: 7.4, 100 mM NaCl, 1% IGEPAL CA-630, 0.1% SDS, 0.5% sodium deoxycholate, 1X cOmplete Protease Inhibitor cocktail (Roche)]. Resuspended pellets were mixed 1:1 with 0.1 mM glass beads (Cole-Parmer) in 2 mL screwcap tubes, and vortexed at max-speed on a Vortex-Genie 2 equipped with a MN bead tube holder (Macherey-Nagel) for 2.5 min at 4 ˚C, after which tubes were rested on ice for 2 min. This bead beating and cooling cycle was repeated 3X. After the final bead beating step, tubes were centrifuged for 2 min at 17,000 x g and 4 ˚C. The supernatants were collected, and lysate protein concentration was determined using the DC II protein kit (BioRad), using BSA to generate the protein standard curve. Lysates were diluted to a final protein concentration of 1 mg/mL in Lysis Buffer, and separated into four separate 1 mL aliquots. RNA ligands were trimmed using RNAse I (ThermoFisher) using either “high” (0.8U) or “low” (0.4U) doses, with both conditions receiving 2 uL of TURBO DNAse (Invitrogen), at 37 ˚C for 3 min with 1100 RPM shaking on a Thermomixer (Thermo Scientific). After this incubation, samples were cooled for 5 min on ice, before centrifugation at 21,000 x g for 10 min at 4 ˚C. Supernatants were then transferred to a Proteus Clarification Mini Spin column (Anatrace), and centrifuged at 13,000 RPM for 1 min at 4 ˚C. Final lysate samples were then applied to Dynabeads Protein G magnetic beads (Thermo) loaded with 4 ug of mouse M2 IgG anti-FLAG monoclonal antibody, or non-specific mouse IgG (Santa Cruz), and allowed to rock overnight at 4 ˚C. The following morning, the beads were dephosphorylated using PNK (NEB) and FastAP Alkaline Phosphatase (ThermoFisher) at 37 ˚C for 40 min with 1100 RPM shaking. After PNK and FastAP treatment, the 3' pre-adenylated L3-IR-App adaptor was ligated onto the bead RNA-protein complexes using T4 RNA ligase I (NEB), PNK, and a final adaptor concentration of 0.1 µM. Beads were incubated at room temperature (23 ˚C) for 75 min, and tubes were flicked every 10 min to ensure mixing of beads with ligation mix. After ligation, unligated adaptors were removed with an enzyme mix of 5' Deadenylase (NEB) and RecJf endonuclease (NEB), incubated at 30 ˚C for 1 hr, then for 30 min at 37 ˚C, shaking at 1100 RPM. RNA-protein complexes were eluted from beads using 1X NuPAGE loading buffer (Thermo) with 0.1 M DTT, and incubated at 70 ˚C for 1 min. Elutions were then loaded into 4-12% NuPAGE Bis-Tris acrylamide gels (Thermo), and run in 1X NuPAGE MOPS running buffer (Thermo) supplemented with NuPAGE antioxidant (Thermo) for 180V until the loading dye front reached the bottom of the gel, and the dye front was excised before transfer. Gels were then transferred to 0.45 µm Protran nitrocellulose membranes (Amersham) in 1X NuPAGE transfer buffer (Thermo), supplemented with 10% Methanol and NuPAGE Antioxidant, at 10 V for 1 hr. Membranes were then imaged using a LICOR Odyssey FC imager, and immediately frozen at -80 ˚C. All three complexes were harvested from the anti-FLAG samples treated with the lowest RNase digestion condition (0.4U) in both biological replicates. RNA ligands were liberated from the VF_2432 complexes using Proteinase K treatment of membrane slices, followed by Phenol:Chloroform extraction. RNAs were then recovered following an overnight sodium acetate precipitation with GlycoBlue (ThermoFisher) and a selected reverse transcription primer, and reverse-transcribed using SuperScript IV (Invitrogen). RNAs were then degraded with NaOH treatment, and cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Circularization of cDNA was conducted using CircLigase II (LGC), after which a further bead cleanup was performed. Phusion HF polymerase (Thermo) was used for amplifying cDNA libraries using P3 and P5 Solexa primers with 22 cycles. Final libraries for two biological replicates were purified using bead cleanup, and submitted for sequencing at the University of Minnesota Genomics Center using an Illumina NextSeq 2000 platform.