TITLE: STRIPE-seq library construction
AUTHORS: Robert Policastro, Gabe Zentner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Prepare Total RNA]
Check RNA quality and concentration on an Agilent TapeStation using a High-Sensitivity RNA ScreenTape.
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.
?. [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.
TEX is magnesium-dependent, so ensure that the RNA storage buffer does not contain EDTA.
?. [Terminator Exonuclease (TEX) Digestion of Uncapped RNA]
Create TEX master mix. Prepare a sufficient volume for the number of reactions to be performed + 1 to account for volume loss during pipetting. ..Vortex to mix and spin down.
[Terminator Exonuclease]
[Terminator Exonuclease Reaction Buffer A]
?. [Terminator Exonuclease (TEX) Digestion of Uncapped RNA]
Incubate the TEX reactions in thermal cycler.  for ..
30 °C
[Hold]
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.
?. [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.
?. [Template Switching Reverse Transcription]
Prepare one RTO annealing mix per sample in 0.2 mL PCR tubes... Each sample should have its own unique barcode..Vortex to mix and spin down.
[Sorbitol/Trehalose Solution]
[Reverse Transcription Oligo (RTO)]
[dNTPs]
[Each]
?. [Template Switching Reverse Transcription]
Add  (from step 3) to  (from step 4.1). Vortex to mix and spin down.
[TEX Reaction]
[RTO Annealing Mixture]
?. [Template Switching Reverse Transcription]
Incubate RTO annealing mixture in thermal cycler....
65 °C
4 °C
[Hold]
?. [Template Switching Reverse Transcription]
Prepare template switching reverse transcription (TSRT) reactions. The process of TSRT enriches for the 5' ends of capped RNA in the final library.
?. [Template Switching Reverse Transcription]
Prepare TSRT reaction master mix (per sample).....Vortex to mix and spin down.
[Betaine]
[5X SuperScript II First Strand Buffer]
[DTT]
[SuperScript II Reverse Transcriptase]
Add reverse transcriptase to master mix just prior to adding to samples.
?. [Template Switching Reverse Transcription]
Add  (from step 5.1) into the  from step 4.3. Vortex to mix and spin down.
[TSRT Master Mix]
[RTO Annealing Reaction]
?. [Template Switching Reverse Transcription]
TSRT.
?. [Template Switching Reverse Transcription]
First half of TSRT reaction...
25 °C
42 °C
Move on to step 6.2 immediately after the end of step 6.1.
?. [Template Switching Reverse Transcription]
Add TSO. Keep the samples in the thermal cycler while adding the TSO..
[TSO]
Move on to step 6.3 immediately after end of step 6.2.
?. [Template Switching Reverse Transcription]
Second half of TSRT reaction....
42 °C
70 °C
[Hold]
This is a good time to prepare the library PCR master mix in step 8.1.
?. [Template Switching Reverse Transcription]
Cleanup of TSRT product.Pipette  up and down 10 times into  from step 6.3.Incubate for  at .Place tubes on magnetic rack and incubate for  at .Carefully aspirate supernatant, leaving ~ in tube to avoid sucking up beads.While tube is still on rack, wash beads with , and immediatly discard wash without incubation.Air dry beads for  at .Resuspend beads in , and incubate on magnetic rack for  at .Transfer  into new 0.2 mL PCR tubes.
[RNAClean XP Beads]
[TSRT Reaction]
0 Room temperature
0 Room temperature
2 µl
[70% Ethanol]
0 Room temperature
[Nuclease Free Water]
0 Room temperature
[Supernatant]
?. [Library PCR]
Prepare library PCR reaction.
?. [Library PCR]
Create library PCR master mix (per sample)....Vortex to mix and spin down.
[2X KAPA HiFi HotStart ReadyMix]
[Forward Library Oligo (FLO)]
[Reverse Library Oligo (RLO)]
?. [Library PCR]
Add  (from step 8.1) into  (from step 7). Vortex to mix and spin down.
[Library PCR Master Mix]
[Cleaned TSRT Product]
?. [Library PCR]
Run library PCR reaction.Initial Denaturation:16-20 cycles:Final Extension:
95 °C
98 °C
63 °C
72 °C
72 °C
[Hold]
?. [Library PCR]
Size selection of final library. SPRI bead size selection is used to remove fragments that are outside the ideal size for Illumina sequencing.
?. [Library PCR]
Removal of small fragments.Pipette  up and down 10 times into  from step 9.Incubate for  at .Place tubes on magnetic rack and incubate for  at .Carefully aspirate supernatant, leaving ~ in tube to avoid sucking up beads.While tube is still on rack, wash beads with  and immediately discard wash without incubation.Air dry beads for  at .Resuspend beads in  and incubate on magnetic rack for  at .Transfer  to new 0.5 mL tube.Optional: Reserve  from beads if you would like to see library size distribution after removing small fragments.
[RNAClean XP Beads]
[Library PCR Product]
0 Room temperature
0 Room temperature
2 µl
[70% Ethanol]
0 Room temperature
[Nuclease Free Water]
0 Room temperature
[Supernatant]
[Remaining Supernatant]
?. [Library PCR]
Removal of large fragments.Pipette  up and down 10 times into  from step 10.1. Make sure to vortex the beads again prior to use.Incubate for  at .Place tubes on magnetic rack and incubate for  at .Transfer  to new tube.Pipette  up and down 10 times into  from previous step.Incubate for  at .Place tubes on magnetic rack and incubate for  at .Carefully aspirate supernatant, leaving ~ in tube to avoid sucking up beads.While tube is still on rack, wash beads with , and immediately discard wash without incubation.Air dry beads for  at .Resuspend beads in , and incubate on magnetic rack for  at .Transfer  to new tube.
[RNAClean XP Beads]
[Cleaned Product]
0 Room temperature
0 Room temperature
[Supernatant]
[RNAClean XP Beads]
[Supernatant]
0 Room temperature
0 Room temperature
2 µl
[70% Ethanol]
0 Room temperature
[Nuclease Free Water]
0 Room temperature
[Supernatant]
?. [Library Quality Control]
Run final libraries on the  Agilent TapeStation using a High Sensitivity D5000 ScreenTape.
Final libraries should be distributed between 250 to 750 bp with a total library amount of 25 to 100 ng.
?. [Terminator Exonuclease (TEX) Digestion of Uncapped RNA]
Prepare TEX reactions in 0.2 mL PCR tubes. Up to .Nuclease free water to  total reaction volume.Vortex to mix and spin down.
[TEX Master Mix]
[Total RNA]
2 µl