Mar 23, 2020

Public workspaceSTRIPE-seq library construction V.3

DOI
dx.doi.org/10.17504/protocols.io.bdtri6m6
  • 1Indiana University
Gabe Zentner
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DOI: dx.doi.org/10.17504/protocols.io.bdtri6m6
Protocol Citation: Robert Policastro, Gabe Zentner 2020. STRIPE-seq library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.bdtri6m6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2020
Last Modified: March 23, 2020
Protocol Integer ID: 34385
Keywords: TSS, transcription, transcription start site
Abstract
Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation; however, current protocols for genome-wide TSS profiling are laborious and expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5’ ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and bead cleanups, a STRIPE-seq library can be constructed in approximately 5 hours.
Materials
MATERIALS
ReagentTerminator 5-Phosphate-Dependent ExonucleaseLucigenCatalog #TER51020
ReagentRNAClean XPBeckman CoulterCatalog #A63987
Reagent5M BetainThermo Fisher ScientificCatalog #AAJ77507UCR
ReagentKAPA HiFi HotStart ReadyMixRocheCatalog #KK2601
ReagentSorbitolDot ScientificCatalog #DSS23080-500
ReagentTrehaloseMP BiomedicalsCatalog #0210309705
ReagentdNTPs 10 μM eachVWR International (Avantor)Catalog #97063-232
ReagentSuperScript II Reverse TranscriptaseThermo Fisher ScientificCatalog #18064014
ReagentRNA ScreenTapeAgilent TechnologiesCatalog #5067-5576
ReagentHigh Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592


Before start
Prepare 3.3 M sorbitol/0.66 M trehalose solution as per Batut and Gingeras (PMID 24510412).

  1. Add Amount2 mL RNase-free H2O to a 50 mL tube.
  2. Add Amount8.02 g trehalose to the tube.
  3. Add Amount3 mL RNase-free H2O .
  4. Add Amount17.8 g sorbitol to the tube.
  5. Add Amount5.5 mL RNase-free H2O
  6. Bring volume to 30 mL with Amount0 mL RNase-free H2O
  7. Transfer to an RNase-free glass bottle and autoclave at 121°C for 30 min.

Store Amount1.5 mL aliquots at TemperatureRoom temperature protected from light.







Prepare Total RNA
Prepare Total RNA
Check RNA quality and concentration on an Agilent TapeStation using a High-Sensitivity RNA ScreenTape.

Expected result
You should have at least 50 to 200 ng of total RNA at a concentration of at least 30 to 125 ng/μl. Your total RNA should also not be highly degraded, as measured by the quality of the rRNA peaks.




Equipment
TapeStation
NAME
Agilent
BRAND
G2991AA
SKU
https://www.agilent.com/en/product/tapestation-automated-electrophoresis/tapestation-instruments/4200-tapestation-system-228263
LINK



15m
Terminator Exonuclease (TEX) Digestion of Uncapped RNA
Terminator Exonuclease (TEX) Digestion of Uncapped RNA
Prepare TEX Reaction. TEX preferentially degrades uncapped RNA, thus reducing the amount of rRNA and degraded mRNA fragments in the sample.
Note
TEX is magnesium-dependent, so ensure that the RNA storage buffer does not contain EDTA.

Create TEX master mix. Prepare a sufficient volume for the number of reactions to be performed + 1 to account for volume loss during pipetting.
  1. Amount0.2 µL Terminator Exonuclease .
  2. Amount0.2 µL Terminator Exonuclease Reaction Buffer A .
Vortex to mix and spin down.
3m
Prepare TEX reactions in 0.2 mL PCR tubes.
  1. Amount0.4 µL TEX Master Mix
  2. Up to Amount1.6 µL Total RNA .
  3. Nuclease free water to Amount2 µL total reaction volume.
Vortex to mix and spin down.
Incubate the TEX reactions in thermal cycler.
  1. Temperature30 °C for Duration01:00:00 .
  2. Temperature4 °C Hold .
Note
This is a good time to prepare the Reverse Transcription Oligo (RTO) annealing and Template Switching Reverse Transcription (TSRT) reaction mixtures from steps 4.1 and 5.1.




1h
Template Switching Reverse Transcription
Template Switching Reverse Transcription
Anneal reverse transcription oligo (RTO) to RNA. STRIPE-seq primes reverse transcription via a random pentamer adjacent to the full length TrueSeq R2 adapter (including the barcode) in the RTO.
Prepare one RTO annealing mix per sample in 0.2 mL PCR tubes.
  1. Amount1.5 µL Sorbitol/Trehalose Solution .
  2. Amount1 µL Reverse Transcription Oligo (RTO) Concentration10 micromolar (µM) . Each sample should have its own unique barcode.
  3. Amount0.5 µL dNTPs Concentration10 Millimolar (mM) Each .
Vortex to mix and spin down.
5m
Add Amount2 µL TEX Reaction (from step 3) to Amount3 µL RTO Annealing Mixture (from step 4.1). Vortex to mix and spin down.
3m
Incubate RTO annealing mixture in thermal cycler.
  1. Temperature65 °C Duration00:05:00 .
  2. Temperature4 °C Duration00:02:00 .
  3. Temperature4 °C Hold .
7m
Prepare template switching reverse transcription (TSRT) reactions. The process of TSRT enriches for the 5' ends of capped RNA in the final library.
Prepare TSRT reaction master mix (per sample).
  1. Amount2 µL Betaine Concentration5 Molarity (M) .
  2. Amount2 µL 5X SuperScript II First Strand Buffer .
  3. Amount0.5 µL DTT Concentration0.1 Molarity (M) .
  4. Amount0.5 µL SuperScript II Reverse Transcriptase .
Vortex to mix and spin down.
Note
Add reverse transcriptase to master mix just prior to adding to samples.

5m
Add Amount5 µL TSRT Master Mix (from step 5.1) into the Amount5 µL RTO Annealing Reaction from step 4.3. Vortex to mix and spin down.



3m
TSRT.


First half of TSRT reaction.
  1. Temperature25 °C Duration00:10:00 .
  2. Temperature42 °C Duration00:05:00 .
Note
Move on to step 6.2 immediately after the end of step 6.1.

25m
Add TSO. Keep the samples in the thermal cycler while adding the TSO.
  1. Amount0.25 µL TSO Concentration400 micromolar (µM) .
  2. Quickly vortex to mix, spin down, and immediately place tubes back in thermal cycler.
Note
Move on to step 6.3 immediately after end of step 6.2.

3m
Second half of TSRT reaction.
  1. Duration00:25:00 Temperature42 °C .
  2. Duration00:10:00 Temperature70 °C .
  3. Temperature4 °C Hold .
Note
This is a good time to prepare the library PCR master mix in step 8.1.

30m
Cleanup of TSRT product.
  1. Transfer the TSRT product from step 6.3 into 0.5 mL tube.
  2. Pipette Amount8 µL RNAClean XP Beads up and down 10 times into Amount10 µL TSRT Reaction from step 6.3.
  3. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  4. Place tubes on magnetic rack and incubate for Duration00:05:00 at TemperatureRoom temperature .
  5. Carefully aspirate supernatant, leaving ~Amount2 µL in tube to avoid sucking up beads.
  6. While tube is still on rack, wash beads with Amount175 µL 70% Ethanol , and immediatly discard wash without incubation.
  7. Air dry beads for Duration00:05:00 at TemperatureRoom temperature .
  8. Resuspend beads in Amount12 µL Nuclease Free Water , and incubate on magnetic rack for Duration00:01:00 at TemperatureRoom temperature .
  9. Transfer Amount11 µL Supernatant into new 0.2 mL PCR tubes.


20m
Library PCR
Library PCR
Prepare library PCR reaction.

Create library PCR master mix (per sample).
  1. Amount12.5 µL 2X KAPA HiFi HotStart ReadyMix .
  2. Amount0.75 µL Forward Library Oligo (FLO) Concentration10 micromolar (µM) .
  3. Amount0.75 µL Reverse Library Oligo (RLO) Concentration10 micromolar (µM) .
Vortex to mix and spin down.
5m
Add Amount14 µL Library PCR Master Mix (from step 8.1) into Amount11 µL Cleaned TSRT Product (from step 7). Vortex to mix and spin down.
2m
Run library PCR reaction.

Initial Denaturation:
  • Temperature95 °C Duration00:03:00
16-20 cycles:
  • Temperature98 °C Duration00:00:20
  • Temperature63 °C Duration00:00:15
  • Temperature72 °C Duration00:00:45
Final Extension:
  • Temperature72 °C Duration00:02:00
  • Temperature4 °C Hold
45m
Size selection of final library. SPRI bead size selection is used to remove fragments that are outside the ideal size for Illumina sequencing.
Removal of small fragments.
  1. Transfer library PCR product from step 9 into 0.5 mL tube.
  2. Pipette Amount16.3 µL RNAClean XP Beads up and down 10 times into Amount25 µL Library PCR Product from step 9.
  3. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  4. Place tubes on magnetic rack and incubate for Duration00:05:00 at TemperatureRoom temperature .
  5. Carefully aspirate supernatant, leaving ~Amount2 µL in tube to avoid sucking up beads.
  6. While tube is still on rack, wash beads with Amount175 µL 70% Ethanol and immediately discard wash without incubation.
  7. Air dry beads for Duration00:05:00 at TemperatureRoom temperature .
  8. Resuspend beads in Amount17 µL Nuclease Free Water and incubate on magnetic rack for Duration00:01:00 at TemperatureRoom temperature .
  9. Transfer Amount15 µL Supernatant to new 0.5 mL tube.
  10. Optional: Reserve Amount1 µL Remaining Supernatant from beads if you would like to see library size distribution after removing small fragments.

20m
Removal of large fragments.
  1. Pipette Amount8.3 µL RNAClean XP Beads up and down 10 times into Amount15 µL Cleaned Product from step 10.1. Make sure to vortex the beads again prior to use.
  2. Incubate for Duration00:10:00 at TemperatureRoom temperature .
  3. Place tubes on magnetic rack and incubate for Duration00:10:00 at TemperatureRoom temperature .
  4. Transfer Amount22 µL Supernatant to new tube.
  5. Pipette Amount22 µL RNAClean XP Beads up and down 10 times into Amount22 µL Supernatant from previous step.
  6. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  7. Place tubes on magnetic rack and incubate for Duration00:05:00 at TemperatureRoom temperature .
  8. Carefully aspirate supernatant, leaving ~Amount2 µL in tube to avoid sucking up beads.
  9. While tube is still on rack, wash beads with Amount175 µL 70% Ethanol , and immediately discard wash without incubation.
  10. Air dry beads for Duration00:05:00 at TemperatureRoom temperature .
  11. Resuspend beads in Amount16 µL Nuclease Free Water , and incubate on magnetic rack for Duration00:01:00 at TemperatureRoom temperature .
  12. Transfer Amount15 µL Supernatant to new tube.
40m
Library Quality Control
Library Quality Control
Run final libraries on the Agilent TapeStation using a High Sensitivity D5000 ScreenTape.
Expected result
Final libraries should be distributed between 250 to 750 bp with a total library amount of 25 to 100 ng.


15m