1. Design and construct gRNA plasmids for targeting the lncRNA of interest.
1.1 Design the paired gRNA sets with an in-house computational program (gp-designer). Typing in the gene symbol into the program, the full-length sequence of the lncRNA will be extracted for constructing (length=30nt, step=1nt) and filtering (GC%=40%~60%) the sequence substrings. The uniquely aligned (human reference genome, hg19) and paired sequences (spacing, 16~20nt) are exported into a excel sheet with indicated RNAfold structure ratio (DotPercent, high value of “DotPercent” from RNAfold suggests a low chance of forming hairpin structure);
1.2 Choose one or several sets of gRNAs targeting specific regions of lncRNA of interest and clone them into the pre-gRNA plasmid (Addgene #109054) with a Gibson assembly approach (NEB, #E2611S).
2. Cell preparation and transfection.
Before CARPID assay, evaluate the specificity of selected gRNA sets by co-expressing them with wild-type CasRx nuclease (Addgene #109049) and determine the knockdown efficacy via quantitative RT-PCR (Figure 3).
2.1 Seed 4×106HEK293T cells in each 10-cm plate one day before transfection. Cells are cultured in DMEM (Life Technologies) supplemented with 10% inactivated FBS (Life Technologies) and 1% penicillin-streptomycin (Life Technologies #15140163) at 37°C with 5% CO2;
2.2 Co-transfect 4μg of gRNA expressing plasmid and 4μg of BASU-dCasRx plasmid with 24 μg of 0.1% PEI for each 10-cm plate;
2.3 At 24 hours post-transfection, change the medium with fresh medium.
3. Biotin labeling
3.1 At 48 hours post-transfection, change the medium with fresh medium containing 200 μM biotin and incubate for 15 mins at 37°C with 5% CO2;
3.2 Replace the medium with 10 ml ice-cold PBS to quench the reaction;
3.3 Wash cells with 10 ml ice-cold PBS at least three times to remove the remaining biotin.
4. Cell lysis
4.1 Add 1 ml ice-cold PBS to the plate and scrape the cells off, transfer them to a 15 ml tube and pellet the cells by centrifugation at 1,200 rpm for 5 mins;
4.2 Completely remove the PBS and resuspend the cell pellet in 1 ml cell lysis buffer;
4.3 After end-over-end rotating in cold room for 10 mins, spin the lysate at 15,000 rpm for 10 min at 4°C to remove the cell debris;
4.4 Transfer the supernatant to a new centrifuge tube and measure the protein concentration with Pierce Protein Quantitation Assay;
4.5 Adjust the volume the lysates to equalize the protein concentration;
4.6 Set aside 50 μl of lysate as Input, and use the remaining lysates for the following immunoprecipitation.
5. Streptavidin immunoprecipitation
5.1 Add 30 μl of MyOne T1 streptavidin beads to the lysate and incubate in cold room for 2 hours with end-over-end rotation;
5.2 Precipitate the streptavidin beads with DynaMag™-2 Magnet stand and remove the supernatant completely;
5.3 Resuspend the beads with 1 ml ice-cold lysis buffer, and perform end-over-end rotation for 5 mins;
5.4 Repeat Step 5.2 and 5.3 twice and place the tube on the DynaMag™-2 Magnet stand.
6. CARPID-Western Blot (CARPID-WB)
6.1 Remove the supernatant completely and add 30 μl elution buffer to heat elute the biotinylated proteins from the streptavidin beads at 95℃ for 10 min and label it as IP;
6.2 Load the Input and IP samples to 4-20% Bis-Tris gel and transfer proteins to a PVDF membrane;
6.3 Block the membrane with BSA blocker for 1 hour at room temperature (RT) and subsequently incubate the blocked membrane with indicated primary antibodies in cold room overnight;
6.4 After primary antibody incubation, wash the membrane with 1× TBST for three times at RT;
6.5 Incubate the membrane with the corresponding secondary antibodies for 1 h at RT;
6.6 Wash the membrane with 1× TBST for three times and apply appropriate amount of ECL substrate to the washed membrane at RT;
6.7 Detect the signal with the Bio-Rad ChemiDoc Imaging System;
6.8 For signal quantification, use ImageJ software (version 1.8.0_172) to quantify and normalize the WB signals (Figure 4).
7. CARPID-Mass Spectrometry (CARPID-MS)
7.1 For CARPID-MS assay (follow step 5.4), wash the streptavidin beads further with 1 ml of 50 mM ammonium bicarbonate (pH 8.0) at 4°C for three times;
7.2 Resuspend the beads in 50 μl Elution buffer I and incubate at 30°C, mixing at 400 rpm for 60 mins and collect the supernatant to a fresh tube;
7.3 Resuspend the beads with 25 μl Elution buffer II twice and combine all three batches of elutes. Protect samples from light during this procedure;
7.4 Add additional 0.25 µg trypsin into the combined elutes and incubate at 37℃ overnight;
7.5 Add 10% formic acid at a ratio of 1:25 (v/v) to stop the digestion;
7.6 Desalt the digested samples with C18 tips following manufacturer’s instruction and reconstitute in 20 μl of 0.1% formic acid;
7.7 Perform the LC-MS/MS analysis using an Easy-nLC 1200 system coupled to a Q Exactive HF mass spectrometry. Inject 6 µl of samples to a reverse phase C18 column (75 μm i.d. × 15 cm, 3 μm particle size, Thermo Scientific #164568) at a flow rate of 250 nl/min. Mobile phase A (0.1% formic acid in ultrapure water) with an eluting buffer as mobile phase B (0.1% formic acid in 80% acetonitrile) are used together to establish a linear 50-min gradient of 7–25% mobile phase B;
7.8 Peptides are ionized by electrospray at 2.3 kV. The mass spectrometer is operated in positive ion mode acquiring at a resolution of 120,000, with a full MS spectrum (m/z = 350-1800) using an automatic gain control (AGC) target of 3×106. The top 12 most intense ions are selected for higher-energy collisional dissociation (HCD) fragmentation (normalized collision energy 27) and MS/MS spectra are generated with an AGC target of 1×105at a resolution of 30,000. The dynamic exclusion time is set to 30 s.
8. LC-MS/MS Data Processing and Analysis
8.1 Analyze all the raw files created by XCalibur (version 4.0.27) software together with the Proteome Discoverer version 2.2 software, against the UniProt human protein database in Sequest HT node.
8.2 Set the precursor and fragment mass tolerances to 10 ppm and 0.02 Da, respectively. The maximum of two missed cleavage sites of trypsin is allowed;
8.3 Set carbamidomethylation (C) as static modification and set oxidation (M) and acetyl (protein N-terminal) as variable modifications. False discovery rate (FDR) of peptide spectrum matches (PSMs) and peptide identification are determined using the Percolator algorithm at 1% based on q-value;
8.4 For label-free quantification (LFQ), use the Minora Feature Detector node in the processing workflow, and use the Precursor Ions Quantifier node and the Feature Mapper node in the consensus workflow;
8.5 Apply enrichment analysis for proteins with more than one peptide detected. Human keratins are included. Normalize the LFQ abundances across the pulldowns and logarithmized. Impute the missing values with values representing the detection limit of the mass spectrometer. Apply the rank product test to determine proteins statistically enriched in the gRNA transfected samples compared to the pre-gRNA controls. Proteins with an adjusted p-value of ≤ 0.05 and ≥ 2-fold change of abundance are considered significant RBPs of the lncRNA of interest (Figure 5).