Name: GSM8111886
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Isolation of total RNA 1. Spin down cells from the transcription run-on reaction at 1,100 g for 5 min at 4 ◦C in a tabletop centrifuge. 2. Quickly resuspend the cell pellet in 500 μL phenol (pH 7.4). 3. Add an equal volume of AES buffer and incubate at 65 ◦C for 5 min. Mix contents on a vortexer for 10 s, once every minute. 4. Incubate on ice for 5 min. 5. Add 200 μL of chloroform, mix contents on a vortexer at maximum speed for 30 s and incubate at room temperature for 2 min. CRITICAL: Chloroform step must be performed in fume hood. 6. Centrifuge at 14,220 g for 5 min at 4 ◦C. 7. Transfer the upper aqueous phase to a new tube. 8. Ethanol precipitate by adding 2 μL GlycoBlue, sodium acetate (final concentration 300 mM), 2.5X volume of 100 % room temperature ethanol, mixing on a vortexer, incubating contents at room temperature for 10 min followed by centrifugation at 14,220 g for 20 min at 4 ◦C. 9. The precipitated RNA forms a gel-like, blue pellet on the side and bottom of the tube. 10. Remove supernatant carefully and rinse the pellet with 750 μL of ice-cold 75 % ethanol. 11. Centrifuge at 14,220 g for 5 min at 4 ◦C. 12. Remove the supernatant carefully and air dry the pellet for 5–10 min. CRITICAL: It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility in the next step. 13. Resuspend the RNA pellet in 100 μL DEPC-H2O and proceed to purification on QIAGEN RNeasy kit as described below (the QIAGEN RNeasy kit serves to exchange the buffer and remove any unincorporated BrUTPs, as these may inhibit binding of labeled RNA during affinity purification). Purification of RNA on the QIAGEN kit The method for purification of RNA on the QIAGEN kit is adapted from the standard protocol described in the QIAGEN handbook. The detailed protocol is as follows: 1. The RNA sample is adjusted to a total volume of 100 μL with DEPC-H2O. 2. Add 350 μL of RLT buffer and mix thoroughly by pipetting up and down. 3. Add 250 μL of 100 % ethanol and mix thoroughly. 4. Transfer the entire sample, approximately 700 μL, to a RNeasy Mini spin column placed in a 2 mL collection tube. 5. Centrifuge at 1,727 g for 30 s at room temperature and discard the flow-through (this step allows binding of RNA to the column). 6. Add 500 μL of RPE buffer to the spin column, centrifuge at 1,727 g for 1 min at room temperature and discard the flow-through. Repeat this step once more (this is the washing step that removes contaminants from the column). 7. Spin at 1,727 g again for 2 min at room temperature to remove any trace of RPE buffer. 8. Remove the spin column from the collection tube. Place into a fresh 1.5 mL microcentrifuge tube and perform elution. 9. Elution is performed in two steps: Step I: Add 50 μL of DEPC-H2O directly onto the matrix of the spin column, rest for 5 min at room temperature, and centrifuge at 160 g for 1 min at room temperature. It is possible that there will be no eluent or very little eluent after the spin. Transfer the eluent, if any, to a new microfuge tube and keep on ice. Step II: Add 50 μL of DEPC-H2O directly onto the matrix of the column, rest for 5 min at room temperature, and centrifuge at 14,220 g for 2 min at room temperature. 10. Combine eluents from steps 9. The total volume of eluent is 100 μL. 11. Measure RNA concentration using the Nanodrop. RNA concentration at this stage is in the range of 100–500 μg. Measuring RNA at this point is to ensure that RNA is not lost during purification on the kit. PAUSE POINT: RNA at this stage can be stored at 80 ◦C for several months. 2.2.4. Affinity purification of nascent RNA 1. Transfer 50 μL of Protein G Dynabeads slurry to a 1.5 mL microcentrifuge tube. 2. Allow magnetic beads to settle by incubating the tube containing beads on magnetic rack for 1 min. 3. Discard the bead storage buffer. 4. Wash beads by resuspending in 0.5 mL TBST, gently shaking on a nutator at 4 ◦C for 3 min, allowing beads to settle on magnetic rack, and then carefully removing the supernatant. 5. Repeat the washing step once more. 6. Resuspend washed beads in 50 μL TBST. 7. Add 2 μg of anti-BrdU monoclonal antibodies to washed beads and gently shake on a nutator for 10 min at room temperature. 8. Add 150 μL of blocking buffer and incubate for 30 min with gentle shaking at room temperature. 9. Capture antibody-bound blocked beads using the magnetic rack and discard the buffer. 10. Wash beads twice with 300 μL of TBSTR as described above in step 4. 11. Resuspend beads in 100 μL TBSTR. 12. Incubate bromo-UMP labelled RNA from section 2.2.3 above at 65 ◦C for 5 min in a water bath (this step is to denature any RNA secondary structure that may reduce the efficiency of RNA binding to the antibody). 13. Keep RNA on ice for 2 min. 14. Mix RNA with affinity beads from step 11 and incubate on a nutator with gentle shaking for 30 min at room temperature. 15. After a brief spin, capture beads on the magnetic rack. 16. Discard the supernatant. 17. Wash beads three times as described above in step 4 with TBSTR on magnetic rack. 18. Resuspend beads with bound RNA in 0.5 mL of TRIzol reagent. 19. Incubate for 5 min at room temperature. 20. Add 100 μL of chloroform to the tube in fume hood. 21. Shake vigorously on a vortexer and incubate the sample at room temperature for 10 min. 22. Centrifuge at 14,220 g for 10 min at 4 ◦C. 23. The upper aqueous phase containing RNA is carefully transferred to a 1.5 mL microcentrifuge tube. CRITICAL: It is important not to take even a trace of the lower, red, organic phase while transferring RNA as it may contain contents that may affect the quality of the GRO-seq library. 24. Ethanol precipitate RNA by adding 2 μL GlycoBlue, sodium acetate (final concentration 300 mM), 2.5X volume of 100 % room temperature ethanol, mixing on a vortexer, and incubating contents at room temperature for 10 min followed by centrifugation at 14,220 g for 20 min at 4 ◦C. CRITICAL: The amount of affinity purified labelled RNA at this stage is very small. Addition of GlycoBlue, which acts as an inert carrier that helps to precipitate small amounts of RNA, maximizes RNA yield, and ensures that RNA pellet is visible after precipitation. 25. The RNA forms a blue pellet. Carefully remove and discard the supernatant. CRITICAL: The RNA pellet is flimsy and easily detaches from the wall of the microcentrifuge tube. Take extreme caution while removing supernatant as it is easy to lose the RNA pellet at this step. 26. Rinse pellet with 1 mL of ice-cold 75 % ethanol. 27. Centrifuge at 14,220 g for 5 min at 4 ◦C. 28. Carefully remove the supernatant. 29. Air-dry the pellet for 5–10 min. 30. Resuspend pellet in 22 μL of DEPC-H2O. CRITICAL: The RNA at this point is extremely low in concentration. Use only 1 μL of the sample to measure concentration by Nanodrop. PAUSE POINT: Affinity purified, nascent RNA at this stage can be stored at 80 ◦C for several months. CRITICAL: Before proceeding with library preparation, it is important to check if there is DNA contamination in the sample by RT-PCR analysis with and without reverse transcriptase. If performing RTPCR, RNA concentration as low as 100 ng per reaction can be used. Preparation of cDNA library. Begin first strand synthesis of RNA-seq library preparation with total volume from step 5 of “rRNA depletion” following the protocol described in NEBNext Ultra II Directional RNA Library Prep Kit for Illumina Instruction section 2.6 First Strand cDNA Synthesis, and described below (https://www.neb. com//media/nebus/files/manuals/manuale7760_e7765.pdf). First strand cDNA synthesis 1. Assemble the first strand synthesis reaction on ice by adding the following components in a total volume of 20 μL: 10 μL of rRNA depleted RNA from the step above, 8 μL of NEBNext Strand Specificity Reagent, 2 μL of NEBNext First Strand Synthesis Enzyme Mix. 2. Mix contents thoroughly by pipetting up and down. 3. Perform first strand cDNA synthesis by incubating the reaction mixture in a thermocycler with the following settings: 10 min at 25 ◦C, 15 min at 42 ◦C, 15 min at 70 ◦C, hold at 4 ◦C. Second strand cDNA synthesis 1. Assemble the second strand synthesis reaction on ice by adding the following components to the first strand synthesis reaction from the step above in a total volume of 80 μL: 8 μL of NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix, 4 μL of NEBNext Second Strand Synthesis Enzyme Mix, 48 μL of nuclease-free water. 2. Keep the tube on ice and thoroughly mix by pipetting. 3. Incubate for 1 h at 16 ◦C in a thermocycler for second strand cDNA synthesis. Purification of double-stranded cDNA using NEBNext Sample Purification Beads 1. Resuspend the NEBNext sample purification beads by agitating on a vortex. 2. To the previously prepared 80 μL second strand cDNA synthesis mix from the step above, add 144 μL of the resuspended beads and mix thoroughly. 3. Incubate at room temperature for 5 min followed by a brief spin to collect contents at the bottom of the tube. 4. Place the tube on a magnetic rack to separate the beads and discard the supernatant. 5. To the magnetic beads, now add 200 μL of 80 % ethanol. 6. Incubate at room temperature for 30 s and discard the supernatant. 7. Repeat steps 5 and 6 once more. 8. Air dry the beads for up to 5 min. CRITICAL: Do not air-dry for longer than 5 min as the DNA recovery is compromised. 9. The tube is removed from the magnetic rack and DNA is eluted from the beads by adding 53 μL of 0.1X TE buffer, thoroughly vortexing, briefly spinning, and incubating at room temperature for 2 min. 10. Place samples on the magnetic rack and transfer 50 μL of the supernatant containing eluted DNA to a PCR tube. PAUSE POINT: The samples may be stored at 20 ◦C at this point. cDNA library end prep 1. To the 50 μL of eluted DNA from the step above, add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL of NEBNext Ultra II Enzyme Mix in a total volume of 60 μL. 2. Mix the contents thoroughly by pipetting. 3. Incubate in a thermocycler with the following settings: 20 ◦C for 30 min, 65 ◦C for 30 min, hold at 4 ◦C. Adaptor ligation 1. Dilute the NEBNext Adaptor in ice-cold Adaptor Dilution Buffer on ice. Dilution depends on the amount of RNA input used for rRNA depletion: 5-fold for 1,000 ng-101 ng RNA input, 25-fold dilution for 100 ng-10 ng RNA input, and 200-fold for 5 ng RNA input. 2. Prepare the ligation reaction by adding the following to the end prep reaction from the step above: 2.5 μL of Diluted Adaptor, 1 μL of NEBNext Ligation Enhancer, and 30 μL of NEBNext Ultra II Ligation Master Mix in a total volume of 93.5 μL. 3. Pipette the mix thoroughly followed by a brief spin to collect residual liquid at the bottom. 4. Incubate the mix at 20 ◦C for 15 min in a thermocycler. 5. To the mix, add 3 μL of USER Enzyme, mix thoroughly by pipetting, and incubate at 37 ◦C for 15 min in a thermocycler. Purification of ligation reaction 1. To the ligation reaction mix, add 87 μL of NEBNext Sample Purification Beads and thoroughly mix by vortexing. 2. Incubate the mix at room temperature for 10 min followed by a brief spin. 3. Place the tube on a magnetic rack and discard the supernatant. 4. Add 200 μL of 80 % ethanol, incubate at room temperature for 30 s, then discard the supernatant. 5. Repeat steps 3 and 4 once more. 6. Briefly spin and place tube back on the magnetic rack, carefully removing the residual ethanol. 7. Air dry the magnetic beads for a maximum of 5 min. CRITICAL: Do not air-dry for longer than 5 min as the DNA recovery is compromised. 8. Remove the tube from the magnetic rack and elute the DNA by addition of 17 μL of 0.1X TE. Mix thoroughly by vortexing followed by a brief spin and incubation at room temperature for 2 min. 9. Place the tube on the magnetic rack and transfer 15 μL of the supernatant to a PCR tube. PAUSE POINT: The samples may be stored at 20 ◦C at this point. PCR enrichment of adaptor ligated DNA 1. The PCR amplification of ligated DNA is performed in a volume of 50 μL. The PCR reaction mix contains 15 μL of adaptor ligated DNA, 25 μL of NEBNext Ultra II Q5 Master Mix, and 10 μL of Index Primer Mix. 2. Thoroughly mix by pipetting followed by a brief spin. 3. Incubate in a thermocycler with the following reaction conditions: 1 cycle of initial denaturation for 30 s at 98 ◦C, 7–16 cycles (adjusted for based upon RNA input) of denaturation and annealing/extension at 98 ◦C for 10 s and 65 ◦C for 75 s, respectively, a final extension for 5 min at 65 ◦C, and hold at 4 ◦C. Recommended PCR cycles are as follows, based on amount of RNA input: 7–8 cycles for 1,000 ng, 11–12 cycles for 100 ng, 14–15 cycles for 10 ng, and 15–16 cycles for 5 ng. Purification of PCR reaction 1. Vortex NEBNext Sample Purification Beads and add 45 μL of resuspended beads to the 50 μL PCR reaction from the step above. 2. Mix thoroughly by vortexing and incubate at room temperature for 5 min. 3. Spin briefly and place the tube on a magnetic rack. Discard the supernatant. 4. Add 200 μL of 80 % ethanol to the tube, incubate at room temperature for 30 s, and discard the supernatant. 5. Repeat steps 3 and 4 once more. 6. Air dry the beads for a maximum of 5 min. CRITICAL: Do not air-dry for longer than 5 min as the DNA recovery is compromised. 7. Remove the tube from the magnetic rack and elute DNA by adding 23 μL of 0.1X TE, mix thoroughly by vortexing, briefly spin, and incubate at room temperature for 2 min. 8. Place the tube on the magnetic rack and transfer 20 μL of supernatant to a new PCR tube. This is the cDNA library/GRO-seq library. PAUSE POINT: The samples may be stored at 20 ◦C at this point. cDNA library assessment cDNA library quality was assessed to verify the library size (200–700 bp peak), purity and quantity using Agilent Tape Station 2200 gDNA-QG protocol following the manufacturer's protocol in the link below and described briefly here: (https://www.agilent.com/cs/library/user manuals/Public/ScreenTape_gDNA_QG.pdf). 1. Prepare sample by mixing 1 μL cDNA library (10–100 ng/μL) from the step above with 10 μL Genomic DNA Sample Buffer. 2. Prepare ladder by mixing 1 μL Genomic DNA Ladder with 10 μL Genomic DNA Sample Buffer. 3. Vortex using IKA vortexer and adaptor at 2000 rpm for 1 min. 4. Briefly spin down to collect samples in the bottom of the tube. 5. Launch the 2200 TapeStation Controller Software and load Genomic DNA ScreenTape device and loading tips into the 2200 TapeStation instrument. 6. Load cDNA library samples and ladders from steps a and b above into the 2200 TapeStation instrument. 7. Select the required samples on the 2200 TapeStation Controller Software. 8. Click Start and specify a file name with which to save your results. CRITICAL: It is important to thaw all samples, bring them to room temperature and mix contents before use. Quantification of cDNA library Quantify libraries using the Qubit assay following Qubit_dsDNA_HS_ Assay_UG protocol following manufacturer's protocol in the link below and described briefly here: (https://tools.thermofisher. com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf). 1. Prepare 200 μL of working solution for each standard and cDNA library sample by diluting the Qubit_dsDNA HS Reagent 1:200 in Qubit_dsDNA HS Buffer, in 0.5 mL plastic PCR tubes. CRITICAL: Do not use glass tubes. 2. To quantify the standard solutions, transfer 190 μL working solution and 10 μL of each standard solution #1 and #2 into a new Qubit 0.5 mL tube. 3. To quantify the libraries, transfer 198 μL of working solution and 2 μL of cDNA library sample into a new Qubit 0.5 mL tube. 4. Mix well, gently, while avoiding bubbling. 5. Briefly spin to get the contents at the bottom of the tube and incubate for 2 min at room temperature. 6. Read sample concentration by selecting dsDNA High Sensitivity as the assay type on the Qubit instrument. CRITICAL: The fluorescence signal is stable for up to 3 h at room temperature and is sensitive to temperature. rRNA removal by QIAseq FastSelect–rRNA Yeast Kit followed by NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina