Low Input NN srna Sequencing

Transcription

1 Adapted from: Williams et al, PNAS 2013;110(11): Zhang et al, Genome Biol. 2013;14(10):R109 Low Input NN srna Sequencing Reagents Mol Bio: Fluor- Plates: (Ambion AM10110) 50% PEG8000 in water (resuspend over night at 37C with shaking) T4 Polynucleotide Kinase (NEB M0201S) T4 RNA Ligase 1 (NEB M0204L) T4 RNA Ligase 2, truncated K227Q mutant (NEB M0351L) GlycoBlue (Ambion AM9516) SuperScript III System (Life ) KAPA HiFi Hot Start 2X PCR Mix (KAPA KK2601) 10bp DNA ladder (Life ) SYBR Gold (Life S ) EasyTides ATP gamma P32; 6000Ci/mmol, 10mCi/ml (Perkin Elmer BLU502Z500UC) BSA Zymo ZR small RNA PAGE recovery Kit (Zymo R1070) MinElute Reaction Cleanup Kit (Qiagen 28204) 19 and 24 nt markers Adenylated 3 adaptors Chemicals: 40% Acrylamide (BioRad 19:1 # ) Urea ultra pure 50% DMSO (aq) 10% APS (<1month old) Ethanol Absolute 0.3M NaCl PEG8000 powder (Sigma) DNA Loading Dye (Orange G) 0.5X TBE Formamide 0.5M EDTA ph8.0 2M Tris- HCl ph7.4 2M MgCl2 2- mercaptoethanol Solutions should be made with ultra pure RNase free water / DEPC- water.

2 Buffers: Made with nuclease free water 10X Yi Buffer for RNA Ligations without ATP Stock chemical: Final concentration: Volume for 5ml: 2M Tris- HCl, ph M 2.5ml 2M MgCl2 0.1M 0.25ml 0.1M DTT 0.01M 0.5ml Water 1.75ml Make ~1ml aliquots and store at - 20C. Denaturing PAA gel solution- prepared volumetrically: 98.8% formamide (1.976ml formamide) 1% v/v EDTA ph 8.0 (20ul 0.5M EDTA) 0.2% bromophenol blue Any safety advice? Aliquote (_ul)? Storage? Or freshly made? Adenylation and purification of 3 Adaptor Notes: - 3 Adaptor sequence must be pre- adenylated to provide the ATP and prevent self- ligation of adaptor during 5 ligation. - Each reaction should contain no more than 1nmol of oligo for adenylation. - I make my own 50% PEG8000 versus alqiuots provided by NEB because it tends to lead to poor adenylation in my hands. - Split the following reaction into 4 (~25ul each) when running them on a gel. 1. Dilute 3 adaptor to 100uM for convenience in the following. Incubate at RT overnight. a. 200um adaptor: 20ul b. NEB 10X T4 RNA Ligase Buffer: 10ul c. 10mM ATP: 5ul d. 50% PEG8000: 40ul e. T4 RNA Ligase 1: 5ul f. Water 20ul 100ul total volume Incubate at room temperature overnight. 2. Run a urea- PAGE gel to purify adenylated adaptor species (20%, 16.5cm x 22cm) a. 21g urea b. 5ml 10X TBE c. 6.25ml water d. 25ml 40% acrylamide (BioRad)

3 Microwave x15sec. swirl to get urea in solution and then cool. Then add the following and cast gel. e. 250ul 10% APS (4C) f ul temed Glass plates have to be thoroughly cleaned. First rinse with water, then clean with 70 % ethanol and Rnase away. Make sure not to touch the side of the plate that will be touching the gel. I pour fresh gels every time. The gels are supposed to be fine for at least a week in the 4 degree but I personally do not use a gel older than 3-4 days for library prep. 2. Mix with 10X Orange G- containing DNA loading dye. Heat x 1min 90C, cool x 3min on ice, and load onto gel. Flush wells with syringe prior to loading! It is advised to load ~3ul of the matching unadenylated 3 adaptor between lanes as a size reference for the adenylation and to approximate adenylation efficiency. dh2o 40 ml Glycerol 30 ml Tris ml EDTA 20 ml Orange G 200 mg 4. Run gel at 450V for ~4 hours (at LEAST until the dye has exited the gel). 150V overnight also works. Running the gel slowly is essential for good separation. 5. Transfer gel to Saran wrap and place on top of a fluorescently- coated TLC plate. Expose to UV light and cut out adenylated adaptor shadows using scalpels. 6. Purify adaptors from gel using the Zymo ZR small- RNA PAGE recovery kit according to manufacturer s instructions. 7. Elute into ultra pure water (sequencing grade) in ~10-15ul. Approximate DNA concentration by NanoDrop. Calculate concentration of each. Hot Labeling of RNA Standards Notes: - RNA standards are used as size references to trace minute RNA quantities throughout sequencing preparation. 19nt and 24nt standards are used (at 200uM stock concentration). - Conversion of 1nmol of each standard is sufficient using 50uCi. Convert each separately. - Perkin Elmer EasyTides gamma P32- ATP is used for convenience of storage. - Use only 80 uci of gamma ATP at a time. This will prevent you from having to run a swipe test after every time you do this. - Marker can be aliquoted and stored diluted. Aliquot should only be used once per experiment.

4 - Alternatively the marker can be directly purified using the Zymo RNA Clean Up Concentrator column instead of by gel purification. a. 200uM RNA marker 5ul b. 10X PNK Buffer (NEB) 1.5ul c. Water 2ul d. Gamma 32P- ATP: 5ul e. PNK 1.5ul 15ul total volume 1. Incubate at 37C x 30min. 2. Pour 15% urea- PAGE gel. Once poured pre- run gel at 600V for >10min. a. 21g urea b. 5ml 10X TBE c. 12.6ml water d ml 40% acrylamide (BioRad) - Once resuspended, 250ul 10% APS and 37.5ul TEMED. 3. Add 15ul of PAA gel loading solution. 4. Run gel at 600V, until BPB is near the bottom of the gel (~1hr) 5. Quickly expose gel to Xray film (1 min) to verify incorporation. 6. Using UV shadowing or overlaying the Xray film on the gel, excise the hot 19 and 24nt bands. 7. Purify using Zymo ZR small RNA PAGE recovery kit. Elute in 10ul each standard, and combine and dilute 1:100 (20ul of hot RNA + 180ul water). 3 Adaptor Ligation to RNA Notes: - Every RNA sample must be ligated independently with a unique 3 pre- adenylated adaptor. Samples will be pooled so it is essential that non- overlapping adaptors are used to distinguish sample reads during sequencing. - All RNA should pass QC (BioAnalyzer, Qubit, etc) before attempting to generate libraries from RNA samples. What does pass mean? For serum and plasma, RNA is too low to be detected by RNA HS Assay (for Qubit), so we Bioanalyzed a random subset to get a rough estimate of the amounts we are dealing with. Maybe Qubit mirna Assay kit can be used to detect amounts lower than 20 ng/ml in future. - All reactions should be performed in siliconized tubes to maximize RNA recovery and prevent nucleic acid adsorption by plastic.

5 - Qiagen mirneasy Micro works well for all samples including fluids. FOLLOW THE PROTOCOL TO THE LETTER TO ENSURE GOOD RESULTS! 1. Provide RNA diluted to a final volume of 8.5ul with water. For the marker- only control reaction, add 2ul of the 1:100 diluted marker to 6.5ul of water. Generally, it is best to have RNA eluted into 10.5ul after extraction. These tubes should be thawed, bioanalyzed or qubit, and then the thawed sample should proceed immediately into this ligation. 3. Make a mastermix of the following ligation components. Vortex hard after the addition of a- d, and then flick vigorously to mix after adding e, f. DO NOT ADD YET. I first make the master mix without the enzymes i.e. RNAseOUT and Ligase. Then I heat shock the samples and add adaptors to each sample and then add the mastermix but just before adding the mastermix to each sample, I add the enzymes to the mastermix. a. Water 2.95ul b. 10X T4 RNA Ligase Buffer- Yi, Minus ATP 2ul c. 50% PEG8000 4ul d. 1:100 diluted radioactive RNA marker 0.05ul e. RNAseOUT 1ul f. T4 Rnl2 trunc K227Q 1ul 11 ul total volume 3. Add the appropriate adenylated adapter at 25uM (1ul for 1.25uM final, or 2 ul for 2.5uM final) to each sample of RNA. (1.25uM is sufficient to ligate ~90% of 100ng total RNA, or up to 400ul of RNA isolated from serum. Further parameters must be characterized.) 4. Heat shock RNA + adapter samples 90C for 1 minute and rest samples on ice 2 mins. 5. Add mastermix to samples from Step 4. Then flick to mix and spin down. Incubate RT 4 hours. 7. Add 3 times the total volume of the combined 3 adapter ligation reactions (excluding the RNA marker- alone reaction). To do this, add 660ul of 100% EtOH to one RNA sample. Then sequentially add 9 additional RNA samples to the ethanol- ligation mixture. This will result in 10 samples of RNA pooled into one tube. Addition of ethanol this way is essential to inactivate the ligase and prevent ligation of adapters to other RNA samples. If 20 samples are being processed you will have

6 2 tubes. ** This is the way published by Tuschl, but we are not sure it is the best way because we are finding a lot of extra adapter in the libraries. 8. Add 20ul of 3M NaCl to each pooled tube that is generated and add 1ul GlycoBlue. Mix well. 9. Precipitate ligations by incubating on ice for 1 hr or dry ice and centrifuge at 4C at max speed for 30mins. 10. Remove supernatant. Spin again at max speed for 10 minutes and remove the residual ethanol. 11. Air- dry pooled pellets for 5 mins. 12. Resuspend each pellet in 10ul of water. (If 20 samples are to be profiled, you will have 20ul of sample from 2 precipitations.) 13. Pool the two 10ul resuspended pellets and add 20ul of PAA gel loading buffer. Incubate at 90C for one minute and ice for 2 minutes. 14. Cast a 15% UREA- PAGE. a. 21g urea b. 5ml 10X TBE c. 12.6ml water d ml 40% acrylamide (BioRad) - Once resuspended, 250ul 10% APS and 37.5ul TEMED. 15. Load the gel. For the samples to be sequenced, divide the volume amongst two adjacent lanes. Run the control marker sample 2-3 lanes adjacent to the sequencing samples. Run gel at 350V for four hours. Load the unligated marker control 2-3 wells away from your actual library. Just on one side is enough to see the shift of the ligated product. 16. Unmount the gel and wrap in Saran wrap. 17. Dip a sharpie in 1:10,000 diluted radiolabeled marker and draw lines around the gel to serve as reference points for excision later. Draw multiple to make identifying the bands to cut out easy. 18. Wrap the gel again in bbbem Saran wrap. With a ruler, then measure the length of 2-3 of the reference lines. 19. Expose the gel in a cassette to a phosphorimager screen for 1-2 hours.

7 21. Excise the gel pieces containing RNA using sterile scalpels. These should correspond to bands at 45bp and 50bp. Which is the size of marker plus the adaptor that it is ligated to, after 3 ligation. Gel- extract using the Zymo ZR kit. Elute the RNA in 9ul total of water. Verify successful isolation by Geiger counting the eluted fraction. Expect 7ul back. 5 Adaptor Ligation to RNA 1. Incubate RNA from step 21 at 90C for 1 minute and then rest on ice for 2 minutes. 2. Prepare a mastermix of the following components. These will be added directly to the 9ul of eluted RNA from Step 21 of the 3 Ligation. a. 10X Yi buffer without ATP 3ul b. 100uM 5 Adaptor (NN or Not) 3ul c. 50% PEG ul d. 10mM ATP 3ul e. T4 RNA LigasE 1 2.5ul 23.5ul total volume 3. Add 2ul of RNA Ligase 1 (NEB) to each tube. Incubate at 37C for 2hours. 4. Cast a 15% Urea- PAGE: a. 21g urea b. 5ml 10X TBE c. 12.6ml water d ml 40% acrylamide (BioRad) - Once resuspended, 250ul 10% APS and 37.5ul TEMED. 4. Add 20ul of PAA gel solution to each sample and incubate for 1min at 90C. Then load samples onto gel. Load the marker- only ligation side by side as a control, and even a 3 ligation reaction for comparison. Run gel at 350V for four hours, or 90V for 14 hours, followed by a faster pulse in the morning. The dye should just leave the gel under these conditions. 5. Use phosphorimager as in Step 20 to image the gel and excise the fragments near the 71 and 76nt marker bands. This may be seen as an upper doublet above a smaller doublet which corresponds to unligated RNAs. 6. After gel excision begin the purify the RNA from the gel using the Zymo ZR Kit, with one modification. After the second heating of the first column, add 1ul of 100uM 3 PCR primer. This will act as carrier and will serve as the RT primer in the next step. 7. Elute RNA in 7ul water.

8 Reverse Transcription Notes: This step is performed in a 1.5ml siliconized eppendorf tube using a heat block, not normal plastic- PCR strips. All reagents for this phase come from the Superscript III RT kit sold by Invitrogen/Life Tech. 1. Denature the eluted RNA at 90C for 1 min. 2. Make the following mix and add 8.7ul to each sample: a. 0.1M DTT 1.5ul b. 5X FS Buffer 3ul c. 10mM DNTP Mix 4.2ul 8.7ul total volume 3. Incubate at 3 minutes for 50C. 4. Add 0.75ul of SSIII to each reaction. Incubate at 42C for 30 minutes. 5. Add 1ul RNaseH to each sample and incubate at 37C for 10 minutes. 6. Add 79ul water to create a 95ul total volume. Library Generation Notes: Kapa 2X HiFi mix used to generate libraries per KAPA recommendations. It is suggested to perform a pilot PCR to optimize cycle number for your sample. i.e., removing aliquots from PCR reaction until bands appear (Steps 1-5). Then, after optimization, generate a large- scale library for further prep and sequencing (Steps 6-18). Cycle titrations- begin at ~17 cycles and go up to 25 if necessary. ** what does a good cycle number look like? What does too many look like? I almost always go with 17, 20 and 23 cycles, the first round. Increase the cycle number if you do not see any bands especially around 100 bp. When the cycle number is too high, the 100 bp band starts to disappear and the products seem to shift upwards in a smear cycles has been working fine for plasma and serum libraries. 1. For the optimization experiment prepare 50ul PCR reactions using the following recipe: a. Water 17ul b. 2X KAPA HiFi Mix 25ul c. 10uM 5 primer 1.5ul d. 10uM 3 primer 1.5ul e. RT product 5ul 50ul total volume

9 2. Run the following thermocycler program on the sample. *Cycle number will depend on your sample and should be determined according to the above experiment C 3 minutes 2. 98C 20 seconds 3. 65C 15 seconds 4. 72C 15 seconds repeat cycles* 5. 72C 1 minute 6. 4C forever 4. Perform Qiagen ERC column cleanup on the DNA. Digest the tests overnight with PmeI at 37C o C to digest the markers away? a. DNA 10ul b. 10X CutSmart Buffer 2.5ul c. Water 11.25ul d. PmeI 1.25ul 5. Analyze PCR reactions by running a 10% native PAGE: a. 12.5ml 40% acrylamide b. 5ml 10X TBE c. 32ml water d. 800ul 10% APS e. 20ul TEMED 4. Run gel at 300V for 4hr. Use Orange G DNA Loading Dye (10X stock). Load 2ul of the 10bp DNA ladder as well. DNA LADDER MUST BE DILUTED IN CUT SMART BUFFER. SALT CONCENTRATIONS MATTER! 5. Stain gel 10 minutes in SYBR Gold diluted 1:10,000 diluted in 0.5X TBE. Visualize by selecting SYBR gold on the gel imager. 6. When the PCR conditions have been defined, repeat the PCR reaction in a large format (3 x50ul PCR reactions): 7. Run the PCR reaction through a MinElute Reaction cleanup column according to manufacturer s instructions. Elute in 15ul of water. Purify 1.5 PCRs per column. 8. Set up a scaled up PmeI digest for overnight at 37C. (3X for in step 7) a. DNA 30ul b. 10X CutSmart Buffer 7.5ul c. Water 33.75ul

10 d. PmeI 3.75ul 9. Run a 10% Native PAGE. For library prep samples, distribute the pooled PCRs over at least 3, usually 4 lanes. This minimizes overloading.. Use Orange G DNA Loading Dye (10X stock). Load 2ul of the 10bp DNA ladder in Cut Smart buffer like in step 4?. It should run for 4 hours at 300V for good separation of 80 and 100 in step 8 below. 8. Excise the band at p, being careful to exclude adapter dimer products at 80bp. 9. Crush gel piece with a p1000 tip in a siliconized 1.5 ml tube 10. Add ~ ul of 1X TEN Buffer to gel piece and vortex for 1 minute. 11. Incubate sample at 37C with constant vortexing for 1 hr then move to overnight at 4C. 12. Remove TEN buffer from gel piece, and to the buffer, add 3 volumes of absolute ethanol. Add 1ul of GlycoBlue for visualization. Freeze on dry ice 30 minutes and then spin at maximum speed for 30 minutes at 4C. 13. Remove supernatant and wash DNA pellet with 1ml 70% EtOH. Spin again for 20 minutes at max speed, 4C. 14. Air dry pellet. 15. Resuspend library pellet in 15ul of EB buffer. 16. Perform Qubit dsdna HS assay to quantitate library concentration 17. Calculate molarity of the library: Assume the average size of the library is 95bp fragments. bp dsdna: 650 g/mol / bp DNA so 95bp: 95 * 650 = 61,750 g/mol = 61,750 ng/nmol Say my library gave 1.68E3 ng/ml by Qubit a. Convert ng/ml to ng/ul: (1.68Eng/ml) x (1ml/1000ul) = 1.68ng/ul b. Determine nmol/ul in library: (1.68ng/ul) / (61,750ng/nmol) = 2.72E- 5 nmol/ul x1000 = 2.72E- 2 pmol/ul=um =0.0272=27.2nM

11 18. Run a High Sensitivity DNA Bioanalyzer chip to verify absence of dimers and good library size quality. ~100 bp, since that is the size that is cut out of the gel 19. Dilute libraries to 10nM and perform additional QC or submit for sequencing. 1nM is the lowest they will take. Quality Control experiments could include qpcr for expected mirnas compared to an old library. Dilute libraries to 0.5 nm to use for qpcr. The sequencing core accepts a range of concentrations from 2nM- 10nM. BioAnalyzer results are required for submission of the library.