TITLE: Minibulk v2 (modified Prime-seq)
AUTHORS: Daniel V Brown

[DESCRIPTION]
Cost-efficient library generation by early barcoding has been central in propelling single-cell RNA sequencing. Here, we optimize and validate prime-seq, an early barcoding bulk RNA-seq method. We show that it performs equivalently to TruSeq, a standard bulk RNA-seq method, but is fourfold more cost-efficient due to almost 50-fold cheaper library costs. We conclude that prime-seq is currently one of the best options to set up an early barcoding bulk RNA-seq protocol from which many labs would profit.

WEHI advanced Genomics facility amendments from Prime-seq:
The plasticware has been changed from 96w plates to 384w and volumes have been reduced 1/4. The reverse transcription of Prime-Seq does not use RNase inhibitors, which I have included.
The primers used for full length cDNA amplification have been taken from 10x Genomics chemistry. This adds compatibility with ONT sequencing (FLT-Seq) developed by Jafar and compatibility with 10x Genomics indexing primers set TT.

[BEFORE_START]
Wipe bench surfaces with PCR clean wipes and keep working environment clean.

[GUIDELINES]
All reagents and plastic-ware can be found in the 'Materials' section.

Use only RNase free supplies and clean all surfaces and tools with RNase Away prior to working
 
Make sure all steps involving cell lysate and RNA before reverse transcription are carried out swiftly and on ice.
 
All primer sequences are listed below:
 
   Oligo Vendor Purification Concentration Sequence       barcoded oligo-dT (E3V6NEXT)


Specific barcoded oligodT (MB2_Kx_DS_RT primer): 

Base design is based off Karst et al., https://doi.org/10.1038/s41592-020-01041-y
MB2 = protocol
KF = Karst design forward UMI = NNNNNNNNYRNNNYRNNN
KR = Karst design reverse UMI = NNNNNNNNRYNNNRYNNN
Forward and reverse designs to improve complexity in Illumina sequencing
DS = David Squire designed the well barcodes and thermodynamic properties
RT = reverse transcription

   



[STEPS]
SECTION: Preparation
1. Clean all surfaces and pipettes with PCR clean wipes
SECTION: Preparation
3. Thaw frozen buffers and primers on ice
SECTION: Preparation
4. Prepare fresh 80% EtOH
6. Input to minibulk v2 is extracted RNA or sorted cells.
It is essential, however, that the samples either have the same input or that they are normalized after the RNA is extracted, otherwise sequencing depth per sample will be impacted.
SECTION: Sample Preparation
7. Normalize the samples so they are all the same concentration. 
 

SECTION: Sample Preparation
8. Transfer 1.2 µL of the normalized RNA to a new plate. Return the unnormalized RNA to the freezer. 

SECTION: Reverse Transcription
10. Prepare Reverse Transcription Mix
Store on ice until use

 

SECTION: Reverse Transcription
13. Add 1.3 µL Reverse Transcription Mix to each well with the FlexDrop

SECTION: Reverse Transcription
14. Incubate for 60 min at 50 °C


SECTION: cDNA Pooling &amp; Purification
16. Pool all wells of each plate by inversion into a SBS reservoir using a centrifuge 400 x g, 2 min, 4 °C 
collect into a 5mL LoBind tube.

SECTION: cDNA Pooling &amp; Purification
19. Incubate for 5 min at 20 Room temperature to allow binding of the cDNA onto beads


SECTION: cDNA Pooling &amp; Purification
20. Wash with 2 mL of 80% EtOH while the tube is on the magnet. Discard the supernatant

 

SECTION: cDNA Pooling &amp; Purification
21. Repeat wash step once more
SECTION: cDNA Pooling &amp; Purification
23. Air dry beads for 2 min

 

SECTION: cDNA Pooling &amp; Purification
24. Elute the beads in 45 µL of UltraPure Water 

SECTION: Full length cDNA Amplification
35. Prepare Pre Amplification Mix

 

SECTION: Full length cDNA Amplification
36. Add 30 µL Pre Amplification Mix to 20 µL cDNA sample

SECTION: Full length cDNA Amplification
37. Incubate the Pre Amplification PCR as follows:


 

 

SECTION: cDNA Bead Purification
38. Mix sample with 40 µL SPRIselect for a 0.8x SPRI to sample ratio

SECTION: cDNA Bead Purification
39. Incubate for 5 min at 20 °C


SECTION: cDNA Bead Purification
40. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Bead Purification
41. Wash with 180 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: cDNA Bead Purification
42. Repeat wash step once more
SECTION: cDNA Bead Purification
43. Air dry beads for 2 min

 

SECTION: cDNA Bead Purification
44. Elute cDNA in 12 µL UltraPure Water

SECTION: cDNA Bead Purification
45. Incubate for 5 min  at RT and transfer 10 µL  to a new PCR tube or plate
 


SECTION: cDNA Quantification and Quality Check
46. Quantify the cDNA using Qubit DNA HS following the manufacturer's protocol. Use 2 μl of clean cDNA for quantification.
SECTION: cDNA Quantification and Quality Check
47. Quality check the cDNA using the Tapestation D5000 tape.

 

 

SECTION: Library Preparation
48. The input to library preparation is amplified full length cDNA from each pooled 384w plate.
 

SECTION: Library Preparation
49. Prepare Fragmentation Mix
Take note of the total cDNA input in ng as it will be relevant in later PCR step
 

 

 

SECTION: Library Preparation
50. Vortex the Fragmentation Mix for5 s and incubate in thermocycler immediately

SECTION: Library Preparation
51. Incubate the Fragmentation reaction in a thermocycler as follows:

 

 

SECTION: Adapter Ligation
52. Prepare Adapter Ligation Mix


 


SECTION: Adapter Ligation
53. Add 6.7 µL  Adapter Ligation Mix to each fragmentation sample
SECTION: Adapter Ligation
54. Incubate for 15 min at 20 °C in a thermocycler
Set heated lid to off

SECTION: Adapter Ligation
55. Add 7.7 µL Buffer EB to Samples
SECTION: Adapter Ligation
56. Mix samples with 16 µL SPRI select beads for a 0.8x SPRI ratio


 


SECTION: Adapter Ligation
57. Incubate for 5 min at 20 °C
SECTION: Adapter Ligation
58. Place the plate on the magnet stand until clear and discard supernatant.   


SECTION: Adapter Ligation
59. Wash with 180 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant
SECTION: Adapter Ligation
60. Repeat wash step once more
SECTION: Adapter Ligation
61. Air dry beads for 2 min

 

SECTION: Adapter Ligation
62. Elute samples in 22 µL water for 5 min off magnet
Incubate 2 min on magent
Transfer 20 µL sample to a new tube

SECTION: Library PCR
64. Add 5 µL of    to each sample.
Take care to change tips and avoid cross contamination

 

SECTION: Library PCR
63. Prepare Library PCR Mix by adding 25 µL  Kappa HiFi polymerase to the 0.2mL tube containing purified adapter ligated sample.

SECTION: Library PCR
65. Incubate the Library PCR reaction as follows:

 


 


 


SECTION: Library Double Size Selection
66. Mix Index PCR with 25 µL SPRI select beads

SECTION: Library Double Size Selection
67. Incubate for 5 min at 20 °C
SECTION: Library Double Size Selection
68. Place the plate on the magnet stand until clear and transfer 75 µL supernatant to clean well.  
 


SECTION: Library Double Size Selection
69. Mix supernatant with 15 µL SPRI select beads
 

SECTION: Library Double Size Selection
70. Incubate for 5 min at 20 °C
SECTION: Library Double Size Selection
71. Place the plate on the magnet stand until clear and discard supernatant.   


SECTION: Library Double Size Selection
72. Wash with 180 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Library Double Size Selection
73. Repeat wash step once more
SECTION: Library Double Size Selection
74. Air dry beads for 2 min

 

SECTION: Library Double Size Selection
75. Elute in 22 µL UltraPure Water. 
SECTION: Library Double Size Selection
76.  Incubate for 5 min and then place on magnet until clear. 
Transfer 20 µL eluted library to new tube. 
 


SECTION: Library QC and quantification
77. Quantify and quality control the library using the Tapestation D1000 kits.


 

SECTION: Sequencing
78. Samples should be submitted according to your Sequencing Facility specifications.

At least 8 cycles are required for the Index Read 1 (i7) 
28 cycles for Read 1 (BC+UMI). 
Read 2 (DNA) should be adjusted based on the quality of the genome being mapped to, but for human and mouse 50 cycles is sufficient.

Some potential sequencing options:

 

SECTION: Reverse Transcription
15. Incubate for 10 min at 80 °C 

SECTION: Preparing lysis buffer
7. Prepare the following lysis buffer to be dispensed by the FlexDrop

 

SECTION: Reverse Transcription
9. Prepare Reverse Transcription Mix
Store on ice until use
 

SECTION: Reverse Transcription
12. Add 1.3 µL Reverse Transcription Mix to each well with the FlexDrop

SECTION: Reverse Transcription
13. Incubate for 60 min at 50 °C


SECTION: Reverse Transcription
14. Incubate for 10 min at 80 °C 

SECTION: cDNA Pooling &amp; Purification
15. Pool all wells of each plate by inversion into a SBS reservoir using a centrifuge 400 x g, 2 min, 4 °C 
collect into a 5mL LoBind tube.

SECTION: cDNA Pooling &amp; Purification
17. Perform 1x SPRI ratio cleanup with SPRI beads diluted 1:4 in SPRI buffer
SECTION: cDNA Pooling &amp; Purification
18. Incubate for 5 min at 20 Room temperature to allow binding of the cDNA onto beads


SECTION: cDNA Pooling &amp; Purification
19. Wash with 2 mL of 80% EtOH while the tube is on the magnet. Discard the supernatant

 

SECTION: cDNA Pooling &amp; Purification
20. Repeat wash step once more
SECTION: cDNA Pooling &amp; Purification
22. Air dry beads for 2 min

 

SECTION: cDNA Pooling &amp; Purification
23. Elute the beads in 45 µL of UltraPure Water 

SECTION: cDNA Pooling &amp; Purification
24. Incubate for 5 min  at RT off the magnet
Incubate for 2 min on the magnet and transfer 43 µL  to a new PCR tube or plate

SECTION: Full length cDNA Amplification
34. Prepare Pre Amplification Mix

 

SECTION: Full length cDNA Amplification
35. Add 30 µL  Pre Amplification Mix to 20 µL  cDNA sample

SECTION: Full length cDNA Amplification
36. Incubate the Pre Amplification PCR as follows:

 
 
 
 

SECTION: cDNA Bead Purification
37. Mix sample with 40 µL SPRIselect for a 0.8x SPRI ratio

SECTION: cDNA Bead Purification
38. Incubate for 5 min at 20 °C


SECTION: cDNA Bead Purification
39. Place the tube on the magnet stand until clear (~5 min) and discard supernatant
SECTION: cDNA Bead Purification
40. Wash with 180 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: cDNA Bead Purification
41. Repeat wash step once more
SECTION: cDNA Bead Purification
42. Air dry beads for 2 min

 

SECTION: cDNA Bead Purification
43. Elute cDNA in 12 µL UltraPure Water

SECTION: cDNA Bead Purification
44. Incubate for 5 min  at RT off magnet
then 2 min  on magnet
Transfer 10 µL  to a new PCR tube or plate
 



SECTION: cDNA quantification and quality check
45. Quantify the cDNA using Qubit DNA HS following the manufacturer's protocol. Use 2 μl of clean cDNA for quantification.
SECTION: cDNA quantification and quality check
46. Quality check the cDNA using the Tapestation D5000 tape.

 

 

SECTION: Preparation
2. Turn on a lab oven to 50 °C  and another to 65 °C 

SECTION: Preparing lysis buffer
8. Dispense 1.2uL lysis buffer to dried minibulk v2 (PRM_0000) primer plates using the FlexDrop
SECTION: Reverse Transcription
10. Remove 384w plates from -80C freezer, thaw on ice and quick spin
SECTION: Reverse Transcription
11. Incubate 65 °C for 5 min 
After this step increase the oven temperature to 80 °C for later use.

SECTION: Reverse Transcription
11. Remove 384w plates from -80C freezer, thaw on ice and quick spin
SECTION: Reverse Transcription
12. Incubate 65 °C for 5 min 
After this step increase the oven to 80 °C for later use

SECTION: Sample Preparation
9. You may store the sample plates at -80 °C at this point

SECTION: cDNA Pooling &amp; Purification
21. Remove tubes from magnet and quick spin in centrifuge.
Use a large centrifuge for 5mL tubes if necessary
SECTION: cDNA Pooling &amp; Purification
22. Remove tubes from magnet and quick spin in centrifuge.
Use a large centrifuge for 5mL tubes if necessary
SECTION: cDNA Pooling &amp; Purification
25. Incubate for 5 min  at RT off the magnet
Incubate for 2 min on the magnet and transfer 43 µL  to a new PCR tube or plate

SECTION: Exonuclease I Treatment
25. Add 5 µL of ExoI Buffer (10x) and 2 µL  µL of Exonuclease I. Incubate as follows:


 

SECTION: Exonuclease I Treatment
26. Add 2 µL of ExoI Buffer (10x) and 1 µL of Exonuclease I. Incubate as follows:


 

SECTION: Exonuclease I Treatment
26. Perform 0.8x SPRI bead size selection with 40 µL  undiluted SPRI beads.

SECTION: Exonuclease I Treatment
27. Incubate for 5 min  at Room temperature to allow binding of the cDNA onto beads

SECTION: Exonuclease I Treatment
28. Place the tube on the magnet stand for 5 min  and discard supernatant

SECTION: Exonuclease I Treatment
29. Wash with 180 µL  of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: Exonuclease I Treatment
30. Repeat wash step once more
SECTION: Exonuclease I Treatment
31. Air dry beads for 2 min 
 


SECTION: Exonuclease I Treatment
32. Elute the beads in 22 µL  of UltraPure Water

SECTION: Exonuclease I Treatment
33. Incubate for 5 min  at RT off magnet
2 min  on magnet then transfer 20 µL to a new PCR tube or plate

SECTION: Exonuclease I Treatment
27. Perform 0.8x SPRI bead size selection with 40 µL  undiluted SPRI beads.

SECTION: Exonuclease I Treatment
28. Incubate for 5 min  at Room temperature to allow binding of the cDNA onto beads

SECTION: Exonuclease I Treatment
29. Place the tube on the magnet stand for 5 min  and discard supernatant

SECTION: Exonuclease I Treatment
30. Wash with 180 µL  of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: Exonuclease I Treatment
31. Repeat wash step once more
SECTION: Exonuclease I Treatment
32. Air dry beads for 2 min 
 


SECTION: Exonuclease I Treatment
33. Elute the beads in 22 µL  of UltraPure Water

SECTION: Exonuclease I Treatment
34. Incubate for 5 min  at RT off magnet
2 min  on magnet then transfer 20 µL to a new PCR tube or plate

SECTION: cDNA Pooling &amp; Purification
16. Measure the approximate volume in each cDNA pooled tube by measuring by pipette
Should be less than 870 µL 

SECTION: Preparation
5. Prepare 900 µL  diluted AMPure RNA XP beads: 1:4 in SPRI buffer per tube
225 µL to 675 µL 

SECTION: cDNA Pooling &amp; Purification
18. Perform 1x SPRI ratio cleanup with SPRI beads diluted 1:4 in SPRI buffer
SECTION: cDNA Pooling &amp; Purification
17. Measure the approximate volume in each cDNA pooled tube by measuring by pipette
Should be less than 870 µL 

SECTION: Bioinformatic preprocessing
79. The fastqs may be preprocessed with your pipeline of choice. I prefer zUMIs where the parameters are:
 

SECTION: Bioinformatic preprocessing
79.1. When I have multiple plates in a run I have the bclconvert script write the index reads to file and concatenate fastqs from all plates into a single file:

 

SECTION: Bioinformatic preprocessing
79.2. I then use the index read as the first part of the well barcode:

 

SECTION: Bioinformatic preprocessing
79.3. You will need to create a well barcode whitelist by concatenating the index reads used to all well barcodes:

 
all_sample_BCs.csv is the barcode_file in the zUMIs yaml