1.1 Design anti-sense oligo probes tiling the ncRNA of interest using the free online Biosearch Technologies' Stellaris FISH Probe Designer
1.2 Probe design parameters: Tile the entire ncRNA from 5’ to 3’; one 20-mer probe every 100 bp of RNA; spacing between probes = 80; target GC% = 45. The program has a maximum target size of 8 kb, so if your target RNA is > 8 kb, break the sequence into segments. Avoid repeats and regions homologous to non-target genomic sequences.
1.3 Order the probes as custom oligos with a 3’-Biotin-TEG modification and HPLC purification.
1.4 Number the probes based on their position along the RNA from 5’ to 3’. Reconstitute each at 100 mM with water (W4502). Prepare two pools of probes, one with 1 mL of each even numbered probe (2, 4, 6, etc.) and the other with 1 mL of each odd numbered probe (1, 3, 5, etc.). Verify concentration by Nanodrop. Store both the pools at -20 °C when not in use.
1.5 Perform experiments in duplicate, one with odd and the other with even probes, to control for non-specific pull-down. Valid ncRNA-dependent signals will be detected with both pools of probes and stand out from nonspecific noise. This “two probe” pool strategy is especially important for ChIRP-Seq experiments.
2.1 Grow cells in tissue culture flasks or dishes (CLS431110) to confluency. You will need a total of 40 million cells, 20 million per ChIRP with both odd and even probe pools.
2.2 Rinse once with HBSS (H6648) and trypsinize T4049).
2.3 Stop trypsinization with complete media and resuspend into single cell suspension.
2.4 Count cells and transfer 20 million cells into two 50 mL tubes
2.5 Pellet cells at 800 RCF for 4 min.
2.6 Aspirate media and resuspend each tube (20 million cells) in 20 mL of PBS (P7059). Note: P7059 is 10X PBS, and must be diluted 1:10 before use.
2.7 Pellet cells at 800 RCF for 4 min.
2.8 Decant PBS, and carefully aspirate the remaining liquid with tube held at an angle to avoid disturbing the cell pellet.
Perform all steps at room temperature (RT).
3.1 Prepare fresh 1% glutaraldehyde (G5882) in PBS just before use. You will need 40 mL per 40 million cells (i.e., 1.6 mL 25% glutaraldehyde stock + 38.4 mL PBS).
3.2 Tap bottom of tubes to dislodge pellets. Resuspend cells initially in a small volume of 1% glutaraldehyde until no chunks are visible then top up to 20 mL. Mix by inverting and cross-link for 10 min on an end-to-end rotator or shaker.
3.3 Quench the cross-linking with 2 mL of 1.25 M glycine (G7403) and continue rotation for 5 min.
3.4 Pellet the cells at 2000 RCF for 5 min at 4 °C.
3.5 Decant supernatant and wash pellet once with 20 mL of chilled PBS, pelleting cells at 2000 RCF for 5 min at 4 °C.
3.6 Decant supernatant and resuspend the washed, cross-linked cell pellet with 1 mL of chilled PBS per 20 million cells and transfer to a tube.
3.7 Pellet cells at 2000 RCF for 3 min at 4 °C. Decant supernatant and remove as much PBS as possible with a pipette tip.
3.8 Flash-freeze the cell pellets in liquid nitrogen and store at -80 °C indefinitely.
4.1. Thaw frozen cell pellets at RT. Tap hard to dislodge the cell pellet.
4.2. Pellet the cells at 2000 RCF for 3 min at 4 °C and discard supernatant. Use a fine pipette tip to remove the last traces of supernatant.
4.3. On an analytical balance, tare the mass of an empty tube identical to the one used to pellet the cells. Weigh each cell pellet and record the weight.
4.4. Just before use, prepare complete Lysis Buffer (10X the mass of cell pellet, e.g. 1 mL for 100 mg) by supplementing with fresh protease inhibitor cocktail (PIC, P8340), AEBSF (A6456) and RNAse inhibitor (R1158). Mix well.
4.5. Resuspend the cell pellets in 10X volume of complete lysis buffer. For small pellets <25 mg, resuspend in 250 µL complete lysis buffer. To make a smooth suspension without clumps, aliquot 500 µL of the suspension into tubes and use a motorized pellet mixer (such as VWR V8185-904) to break up the clumps. Proceed immediately to sonication.
5.1. Aliquot the cell lysate into 15 mL polystyrene tubes (Falcon 352099). Use < 1.5 mL lysate in each 15 mL tube.
5.2. Sonicate the cell lysate with a Bioruptor (Diagenode) in a cold room using the following parameters: H- high setting, pulse interval- 30 sec ON and 45 sec OFF, cycle time- 15 min each. Change ice in water bath chamber after each cycle. Optimize the sonication time (number of cycles required) until the cell lysate is no longer turbid and the sonicated DNA size range is 100-500 bp, usually 9 cycles ~2 hours. After 5 cycles, pool the sonicated chromatin, mix by pipetting and re-aliquot into 15 mL tubes and continue sonication to ensure homogenous shearing cross all tubes.
Note: Glutaraldehyde cross-linked cells take a significantly longer time to sonicate than formaldehyde cross-linked cells.
5.3. After the lysate is clear, transfer 5 µL lysate into a fresh Eppendorf tube to evaluate shearing. Add 90 µL Proteinase K buffer and 5 µL Proteinase K (P4850). Vortex to mix and incubate at 50 °C for 45 min.
5.4. Purify sonicated DNA from step 5.3 using the ChIP DNA Clean and concentrator kit (Zymo Research D5205). Elute DNA in 30 µL Zymo Elution buffer and check the DNA size on a 1% agarose gel (P5472). If bulk of the sonicated DNA smear is between 100-500 bp, sonication is complete. If not, continue sonication for additional cycles as needed.
5.5. Centrifuge the remaining sonicated chromatin from step 5.2 at 16100 RCF for 10 min at 4 °C. Combine supernatants and proceed with ChIRP setup or aliquot into 1 mL samples and flash-freeze in liquid nitrogen. Store at -80 °C.
6.1. If frozen, thaw sonicated chromatin at RT.
6.2. Remove a 10 µL aliquot for RNA INPUT and another 10 µL for DNA INPUT and keep on ice until ChIRP is completed.
6.3. Prepare 4 mL Hybridization buffer for each pair (odd & even) of ChIRP reactions. Vortex to mix.
6.4. Transfer 1 mL chromatin into two separate 15 mL tubes, one for ChIRP with odd and the other with even probes, and add 2 mL Hybridization buffer to both.
6.5. Thaw ChIRP probes at RT. Add 1 µL of 100 µM (i.e. 100 pmol) odd or even probe pool to separate 1 mL aliquots of chromatin. Mix well and incubate at 37 °C for 4 hours to overnight with rotation in a hybridization oven.
6.6. Twenty minutes before the hybridization is complete, prepare the Streptavidin magnetic C1 beads (Life Tech. 65001, stored at 4 °C). Use 100 µL beads per 100 pmol of probes. Wash 2 x 100 µL beads 3 times with 1 mL unsupplemented lysis buffer, using a magnetic stand to separate the beads from buffer.
6.7. Resuspend the beads in their original volume (100 µL) of lysis buffer, freshly supplemented with 1 µL each of 0.1M AEBSF (A6456), PIC (P8340) and RNase inhibitor (R1158).
6.8. After the hybridization reaction is complete, add 100 µL washed beads to each ChIRP reaction. Mix well and incubate at 37 °C for 30 min with rotation in the hybridization oven.
6.9. Prepare wash buffer (5 mL per ChIRP reaction,. Vortex to mix. Pre-warm to 37 °C. Add 0.01x volume of 0.1M AEBSF (A6456), just before use.
6.10. Wash the beads 5 times with 1 mL of wash buffer at 37 °C. For the first wash, remove lysate on a magnetic stand, resuspend beads in 1 mL of wash buffer, and transfer to an Eppendorf tube. Incubate at 37 °C with rotation for 5 min.
6.11. For subsequent washes, spin the tubes briefly, and separate the beads on a magnetic stand. Remove wash buffer, resuspend in a fresh 1 mL of wash buffer, and incubate at 37 °C with rotation for 5 min.
6.12. With the last wash, resuspend the beads thoroughly. Remove 100 µL for RNA isolation. The remaining 900 µL is for DNA purification. After removing the wash buffer from both, spin the tubes briefly and return them to the magnetic stand and remove the last traces of wash buffer with a 10 µL pipette tip.
7.1. Resuspend the 100 µL bead sample reserved above for RNA (6.12) in 95 µL of Proteinase K buffer.
7.2. Add 85 µL of Proteinase K buffer to the 10 µL RNA INPUT aliquot reserved above (6.2).
7.3. Add 5 µL Proteinase K (P4850) to both the beads (7.1) and the RNA INPUT (7.2) and incubate at 50 °C for 45 min with end-to-end shaking in a hybridization oven.
7.4. Spin the tubes briefly and incubate at 95 °C for 10 min, slide 1.5 mL tube holders (Z708372) to secure the lids before placing in the heat block.
7.5. Chill the samples on ice for 5 min. Add 500 µL TRI Reagent (T9424) and vortex vigorously for 10 sec. Incubate at RT for 10 min. Store at -80 °C or proceed to step 7.6.
7.6. Add 100 µL Chloroform (C2432), vortex vigorously for 10 sec, and spin at 16100 RCF for 15 min at 4 °C.
7.7. Carefully transfer approximately 400 µL of the aqueous phase, avoiding the interface and organic layer (pink), into a fresh Eppendorf tube.
7.8. Add 600 µL 100% Ethanol (459844) & and mix well. Purify RNA using a Norgen total RNA purification kit (37500) by spinning the samples through the spin columns (700 µL the first time followed by the remainder). Wash and elute according to the manufacturer’s instructions. Elute RNA with 30 µL of Elution solution.
7.9. Treat the purified RNA with DNA-free (Life Tech AM1906) per manufacturer’s protocol. After the reaction is complete, heat the samples for 15 min at 65 °C to completely inactivate any remaining DNase.
7.10. Use 1 µL of purified RNA for each qRT-PCR analysis to confirm lncRNA retrieval. GAPDH primers may be used as a negative control.
7.11. Store remaining RNA at -80 °C.
8.1. Prepare 1 mL DNA Elution buffer for each pair of ChIRPs plus DNA INPUT.
8.2. Add 10 µL RNase A (10 mg/mL, R4642) and 10 µL RNase H (10 U/µL, Epicenter R0601K) per 1 mL of DNA Elution buffer just before use. Vortex to mix.
8.3. Resuspend beads reserved for DNA from step 6.12 in 150 µL of DNA Elution buffer supplemented with RNases, dilute 10 µL DNA INPUT from step 6.2 in 140 µL of the same, and incubate all at 37 °C for 30 min with shaking.
8.4. Place the tubes containing beads on a magnetic stand for 1 min and transfer the supernatants to fresh Eppendorf tubes.
8.5. Repeat the DNA Elution for a second time with another 150 µL of DNA Elution buffer plus RNases (8.3 to 8.4), and transfer to the same tube as the first elution (total ~300 µL).
8.6. Add 15 µL Proteinase K (P4850) to each sample and incubate at 50 °C for 45 min with shaking.
8.7. Purify DNA samples using the ChIP DNA Clean and concentrator kit (Zymo Research D5205) per manufacturer’s protocol. Elute DNA in 30 µL Zymo Research DNA Elution buffer.
8.8. Use 2 µL of each purified DNA for qPCR analysis using positive and negative control primers or proceed for high-throughput sequencing library preparation per manufacturer’s (Illumina or NEB) protocol.
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