TITLE: Barcoded Calling Cards and Transcriptomes: Library Preparation
AUTHORS: Matthew Lalli, Urvashi  Thopte, Arnav Moudgil, Michael N. Wilkinson, Xuhua Chen, Robi D. Mitra

[DESCRIPTION]
This protocol is a modification of Moudgil et al's Bulk Calling Cards Library Preparation that uses barcoded self-reporting transposons for Calling Cards and barcoded oligos for transcriptome capture. This protocol describes how to create next-generation sequencing libraries from barcoded self-reporting transposon (SRT) Calling Cards experiments to measure transcription factor binding and gene expression in parallel from collected RNA. 

This protocol assumes you have successfully transformed cells with barcoded piggyBac self-reporting transposons and either undirected piggyBac transposase or a gene expression regulator (GER) fused to piggyBac. Your cells are now ready for RNA extraction, SRT and transcriptome amplification, and library preparation. 

[BEFORE_START]
Please read and familiarize yourself with the manuals for the DirectZol RNA Mini Kit and the Nextera XT Tagmentation Kit. The instructions are meant to summarize those workflows; however, when in doubt, please refer to the manufacturer's instructions for guidance.


[GUIDELINES]
Please read this protocol in its entirety before starting. For several steps, it may help to pre-program your thermocycler with the listed settings. Please also read the BRB-seq paper , especially the Maxima H protocol implementation, to become familiarized with the barcoded transcriptome capture.

[STEPS]
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
1. Harvest cells.  Do not overload columns. If you have more than 107 cells, split cells in half and process on two columns, then merge the RNA pools. 
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
2. Add TRI Reagent directly to cells in a dish. Use the following table as a guide.
 
Lyse the cells and transfer into 1.7 mL tube.

SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
3. Add an equal volume ethanol (95-100%). Homogenize the lysate by vortexing briefly.
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
4. Transfer 700 ul of this mixture into a Zymo-Spin IICR Column spin column placed in a 2 ml collection tube. Centrifuge for 30 seconds at ≥ 8,000g.  Ensure no liquid remains on the column membrane. Repeat centrifugation for samples > 700 µl. 
 

SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
5. Add 400 µL RNA PreWash buffer to column and centrifuge. Discard the flow-through.
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
6. Add 700 µl RNA Wash Buffer to the column and centrifuge briefly. Discard flow-through and centrifuge for 2 minutes. Transfer column into an RNase-free tube.
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
7. Elute RNA by adding 50 µl DNase/RNase-free water or te buffer to column and centrifuging. 
SECTION: RNA Extraction wtih Direct-Zol RNA Miniprep
8. Measure RNA concentration using Nanodrop or Qubit. 
SECTION: First Strand Synthesis (reverse transcription)
9. For first strand synthesis by reverse transcription, process samples according to experimental design. Use a unique BRB-seq primer for each transcriptome to be captured. 

Prepare the reverse transcription (RT) reaction mix:
2 µg total RNA
2 µl of 25 µM BRB-seq_dT30VN primer (e.g., pSeq1-BC1-UMI-dtVN)
1 µl of 10 mM dNTPs
Raise to 14 µl with ddH2O

Incubate RT mix at 65 °Cfor 5 min

Place on ice for 1 minute.
SECTION: First Strand Synthesis (reverse transcription)
10. Create 1x Maxima RT buffer:
For 5 or fewer samples, combine 1 µL of 5X Maxima RT buffer with 4 µL of ddH2O.
Mix by pipetting and store on ice.
SECTION: First Strand Synthesis (reverse transcription)
11. Create a 0.5x Maxima RT H Minus enzyme dilution:
Mix an equal volume of Maxima RT H Minus Enzyme with the 1x Maxima RT buffer made in previous step (e.g. 2 µL of Enzyme + 2 µL of 1x buffer).

You will need 1 µL of the 0.5x enzyme dilution for every sample being processed. Avoid pipetting volumes < 1 µL.
SECTION: First Strand Synthesis (reverse transcription)
12. Add the following to the RT mix:
4 µl 5X Maxima RT Buffer
1 µl RNasin RNase Inhibitor 
1 µl of 0.5X Maxima RT H Minus enzyme (1:1 mixture of 1X Maxima RT Buffer and Maxima RT H Minus enzyme = 100 U)
1 uL SMART_TSO (25 µM)

Mix by pipetteing and incubate at 50 °Cfor 60 min
SECTION: First Strand Synthesis (reverse transcription)
13. Heat inactivate the reaction by incubating at 85 °Cfor 10 min
SECTION: First Strand Synthesis (reverse transcription)
14. Column purify using NucleoSpin Gel and PCR Clean-up kit to remove carry-over oligoDT primers.
 


If pooling, combine 3-5 µl of each sample before column purification. 

       Bring total volume of pooled or individual samples to 100 µL by adding water.  
       Add 200 µl NT1 and proceed with column purification according to manufacturer's instructions.

       Elute in 30-50 µl DNAse/RNAse free water. 
SECTION: First Strand Synthesis (reverse transcription)
15. cDNA can be stored at –20 ºC for long term, or 4 ºC until downstream processing 
SECTION: Amplification of Self-Reporting Transcripts
16. This PCR will specifically amplify self-reporting transcripts from cDNA libraries. Prepare the following solution:

25 µl 2X Kapa HiFi HotStart ReadyMix
1 µl of 25 µM Reverse Primer (partial seq1)
4 µl of purified cDNA* 
19 µl of ddH2O
1 µl of Forward Primer, either:
        25 µM SRT_PAC_F1 primer, if using PB-SRT-Puro
        25 µM SRT_tdTomato_F1, if using PB-SRT-tdTomato

 

SECTION: Amplification of Self-Reporting Transcripts
17. Perform PCR using the following thermocycling parameters:

95ºC for 3 minutes
20 cycles of:
    98ºC for 20 seconds
    65ºC for 30 seconds  
    72ºC for 5 minutes
72ºC for 10 minutes
4ºC forever
SECTION: Purification of PCR Products
20. Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.
SECTION: Purification of PCR Products
21. Add 30 µl beads to each 50 µl PCR mixture (0.6x ratio). Mix by pipetting 10 times until evenly dispersed.
SECTION: Purification of PCR Products
22. Incubate at room temperature for 5 min
SECTION: Purification of PCR Products
23. Place on a magnetic rack for 2 minutes. Aspirate supernatant.
SECTION: Purification of PCR Products
24. Add 200 µl of freshly-prepared 70% ethanol off the magnetic rack and mix. Place on magnetic rack and incubate ≥ 30 seconds. Aspirate ethanol. 
SECTION: Purification of PCR Products
25. Repeat Step 24 on the magnetic rack.


SECTION: Purification of PCR Products
26. Air dry the beads at room temperature for 2 minutes. Do not over dry. 
SECTION: Purification of PCR Products
27. Remove the tube from the magnetic rack. Add 20 µl ddH2O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes. 
SECTION: Purification of PCR Products
28. Place on magnetic rack for 1 minute, or until supernatant is clear.
SECTION: Purification of PCR Products
29. Transfer supernatant to new tube. Quantify product on Tapestation D5000.  
 

SECTION: Generation of Bulk Calling Card Libraries
30. The tagmentation protocol fragments the long PCR products into libraries suitable for sequencing. 

Preheat thermocycler to 55 °C
SECTION: Generation of Bulk Calling Card Libraries
31. Take 1 ng of PCR product and resuspend in a total of 5 µl ddH2O in a PCR strip tube.

 


SECTION: Generation of Bulk Calling Card Libraries
32. Add 10 µl of Nextera Tagment DNA (TD) Buffer and 5 µl of Amplicon Tagment Mix (ATM). Pipette to mix and briefly spin down; bubbles are normal. Incubate at 55 °Cfor 5 min
SECTION: Generation of Bulk Calling Card Libraries
33. Add 5 µl of Neutralization Tagment (NT) Buffer. Pipette to mix and briefly spin down; bubbles are normal. Incubate at room temperature for 5 min
SECTION: Generation of Bulk Calling Card Libraries
34. For SRT libraries, add the following to each PCR tube in order:
15 µl Nextera PCR Mix (NPM)
8 µl ddH2O
1 µl of 10 µM barcoded piggyBac primer (e.g., P5_BC_SRT_STAGGER1)
1 µl of 10 µM indexed Nextera N7 primer (e.g. Nextera_N701)




 

SECTION: Generation of Bulk Calling Card Libraries
35. Perform PCR using the following thermocycling parameters:
72ºC for 3 minutes
95ºC for 30 seconds
16 cycles of:
    95ºC for 10 seconds
    52ºC for 30 seconds
    72ºC for 30 seconds
72ºC for 5 minutes
4ºC forever


SECTION: Purification of Sequencing Libaries
42. Purify PCR libraries using AMPure XP beads. Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.

SECTION: Purification of Sequencing Libaries
43. Add 35 µl beads to each 50 µl PCR mixture (0.7x ratio). Mix by pipetting 10 times until evenly dispersed.
SECTION: Purification of Sequencing Libaries
44. Incubate at room temperature for 5 min
SECTION: Purification of Sequencing Libaries
45. Place on a magnetic rack for 2 minutes. Aspirate supernatant.
SECTION: Purification of Sequencing Libaries
46. Add 200 µl of freshly-prepared 70% ethanol off the magnetic rack and mix. Place on magnetic rack and incubate ≥ 30 seconds. Aspirate ethanol. 
SECTION: Purification of Sequencing Libaries
47. Repeat Step #46 on the magnetic rack.
SECTION: Purification of Sequencing Libaries
48. Air dry the pellet at room temperature for 2 minutes.
SECTION: Purification of Sequencing Libaries
49. Remove the tube from the magnetic rack. Add 11 µl ddH2O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes.
SECTION: Purification of Sequencing Libaries
50. Place on magnetic rack for 1 minute, or until supernatant is clear. Transfer supernatant to new tube.
SECTION: Final Quantitation and Sequencing
51. Measure library concentration on a TapeStation device with a High Sensitivity D1000 ScreenTape. Libraries should be smoothly distributed between 300-600 bp.
 

SECTION: Final Quantitation and Sequencing
52. Libraries can be sequenced on any Illumina sequencing platform. 
 

 

SECTION: Amplification of Full-length Barcoded Transcriptomes
18. If desired, this PCR will amplify full-length transcriptomes from cDNA libraries. Prepare the following solution:

25 µl 2X Kapa HiFi HotStart ReadyMix
1 µl of 25 µM Reverse Primer (partial seq1)
1 ul of 25 uM Forward Primer (SMART)
4 µl of pooled and purified cDNA
19 µl of ddH2O
SECTION: Amplification of Full-length Barcoded Transcriptomes
19. Perform PCR using the following thermocycling parameters:

95ºC for 3 minutes
10 cycles of:
    98ºC for 20 seconds
    60ºC for 30 seconds
    72ºC for 6 minutes
72ºC for 10 minutes
4ºC forever
SECTION: Generation of BRB-seq Libraries
41. Perform PCR using the following thermocycling parameters:

72ºC for 3 minutes
95ºC for 30 seconds
16 cycles of:
    95ºC for 10 seconds
    55ºC for 30 seconds
    72ºC for 30 seconds
72ºC for 5 minutes
4ºC forever

SECTION: Generation of BRB-seq Libraries
40. For BRB-seq library amplification, add the following to each PCR tube in order:
15 µl Nextera PCR Mix (NPM)
8 µl ddH2O
1 µl of 10 µM P5-index-seq1 primer
1 µl of 10 µM indexed Nextera N7 primer (e.g. Nextera_N701)

SECTION: Generation of BRB-seq Libraries
39. Add 5 µl of Neutralization Tagment (NT) Buffer. Pipette to mix and briefly spin down; bubbles are normal. Incubate at room temperature for 5 min
SECTION: Generation of BRB-seq Libraries
38. Add 10 µl of Nextera Tagment DNA (TD) Buffer and 5 µl of Amplicon Tagment Mix (ATM). Pipette to mix and briefly spin down; bubbles are normal. Incubate at 55 °Cfor 5 min
SECTION: Generation of BRB-seq Libraries
37. Take 1 ng of PCR product and resuspend in a total of 5 µl ddH2O in a PCR strip tube.
SECTION: Generation of BRB-seq Libraries
36. In parallel with Calling Card libraries, prepare BRB-seq libaries with tagmentation. 

Preheat thermocycler to 55 °C