TITLE: prime-seq
AUTHORS: Aleksandar   Janjic, Lucas Esteban  Wange, Johannes JWB Bagnoli, Johanna  Geuder, Phong  Nguyen, Daniel  Richter, Christoph  Ziegenhain, Wolfgang Enard, Beate  Vieth

[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 also validate a direct RNA isolation step, show that intronic reads are derived from RNA, and compare cost-efficiencies of available protocols. 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.

[BEFORE_START]
Wipe bench surfaces with RNAse Away 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 (E3V7NEXT) sequences:
 
  
  


[STEPS]
SECTION: Preparation
1. Clean all surfaces and pipettes with RNase Away
SECTION: Preparation
2. Thaw frozen buffers and primers on ice
SECTION: Preparation
3. Prepare 80% EtOH (approximately 45 mL for 96 samples)
SECTION: Prepare Cleanup Beads (22% PEG)
102. Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube

 

 


SECTION: Prepare Cleanup Beads (22% PEG)
103. Incubate at 40 °C and vortex regularly until PEG is completely dissolved
SECTION: Prepare Cleanup Beads (22% PEG)
104. Resuspend Sera-Mag Speed Beads carefully and pipette 1000 µL of bead suspension into a 1.5 mL tube
SECTION: Prepare Cleanup Beads (22% PEG)
105. Place on magnet stand and remove storage buffer

SECTION: Prepare Cleanup Beads (22% PEG)
106. Add 1000 µL of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads



SECTION: Prepare Cleanup Beads (22% PEG)
107. Place on magnet stand and remove supernatant 
SECTION: Prepare Cleanup Beads (22% PEG)
108. Repeat wash step one more time 
SECTION: Prepare Cleanup Beads (22% PEG)
109. Add 900 µL TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads

SECTION: Prepare Cleanup Beads (22% PEG)
110. Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
 

SECTION: Lysate vs Extracted RNA
5. prime-seq can be used on lysate or extracted RNA. 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. Based on your starting material, please follow one of the following cases:
SECTION: Prepare Bead Binding Buffer
111. Prepare Bead Binding Buffer (2x)

 

 


SECTION: Sample Collection
7. Prepare Lysis Buffer according to the number of samples.

 

 



SECTION: Sample Collection
8. Add100 µL of Lysis Buffer to each well of a semi-skirted 96-well PCR plate
SECTION: Sample Collection
9. Add cells or tissue to wells

 

 

SECTION: Sample Collection
10. Transfer 50 µL of lysate to a new plate, return one plate immediately to -80 C freezer to save as a backup

 

SECTION: Proteinase K Digest
11. Add 1 µL Proteinase K (20 mg/mL) and 1 µL EDTA (25 mM) to each well



SECTION: Proteinase K Digest
12. Incubate for 15 min at 50 °C and then heat inactivate the Proteinase K for 10 min at 75 °C




SECTION: Bead Clean Up
13. Mix each bulk sample (50 µL per well) with 100 µL of Cleanup Beads (22% PEG)
SECTION: Bead Clean Up
14. Incubate for 5 min at 20 °C
 

SECTION: Bead Clean Up
15. Place on magnet stand until clear (~3 min) and then discard supernatant
SECTION: Bead Clean Up
16. Wash with 100 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant


 


SECTION: Bead Clean Up
17. Repeat wash step once more


SECTION: Bead Clean Up
18. Air dry beads for 3 min

 



SECTION: DNAse I Digest
19. Add 5 µL H2O and resuspend beads by vortexing vigorously

 

SECTION: DNAse I Digest
20. Prepare DNase I Mix 

 

 
 
SECTION: DNAse I Digest
21. Add  15 µL of DNase I Mix and mix by pipetting



SECTION: DNAse I Digest
22. Incubate DNase I Mix and beads for 10 min at 20 °C


SECTION: DNAse I Digest
23. Heat inactivate the DNase I by adding 1 µL of EDTA (100 mM) and incubating for 5 min at 65 °C



SECTION: DNAse I Digest
24. Place plate on magnet stand until clear (~3 min) and discard the supernatant.
SECTION: DNAse I Digest
25. Wash with 100 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant
SECTION: DNAse I Digest
26. Repeat wash step once more
SECTION: DNAse I Digest
27. Air dry beads for 5 min

 

 

SECTION: Reverse Transcription
28. Prepare Reverse Transcription Mix

 
 

 
SECTION: Reverse Transcription
29. Add 4 µL H2O to the beads
 

SECTION: Reverse Transcription
30. Add 5 µL Reverse Transcription Mix

SECTION: Reverse Transcription
31. Add 1 µL of Barcoded oligodT (E3V7NEXT) (10 µM) per well

SECTION: Reverse Transcription
32. Incubate for 90 min at 42 °C


SECTION: cDNA Pooling & Purification
33. Place the plate on a magnet
SECTION: cDNA Pooling & Purification
34. Pool the supernatant of all wells into a 2 mL tube

SECTION: cDNA Pooling & Purification
35. Add 10 µL of Cleanup Beads (22% PEG) per sample for a 1:1 ratio (e.g. 240 µL for 24 samples)

 

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


SECTION: cDNA Pooling & Purification
37. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Pooling & Purification
38. Wash with 1 mL of 80% EtOH while the tube is on the magnet, discard the supernatant

 

SECTION: cDNA Pooling & Purification
39. Repeat wash step once more
SECTION: cDNA Pooling & Purification
40. Air dry beads for 5 min

 

SECTION: cDNA Pooling & Purification
41. Elute the beads in 17 µL of UltraPure Water 

SECTION: cDNA Pooling & Purification
42. Incubate for 5 min  at RT and transfer to a new PCR tube or plate

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

 

SECTION: Exonuclease I Treatment
44. Mix each sample (20 µL per well) with 16 µL of Cleanup Beads (22% PEG) for a 1:0.8 ratio
SECTION: Exonuclease I Treatment
45. Incubate for 5 min at 20 Room temperature to allow binding of the cDNA onto beads


SECTION: Exonuclease I Treatment
46. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: Exonuclease I Treatment
47. Wash with 50 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: Exonuclease I Treatment
48. Repeat wash step once more
SECTION: Exonuclease I Treatment
49. Air dry beads for 5 min

 

SECTION: Exonuclease I Treatment
50. Elute the beads in 20 µL of UltraPure Water 

SECTION: Exonuclease I Treatment
51. Incubate for 5 min  at RT and transfer to a new PCR tube or plate
SECTION: Full length cDNA Amplification
52. Prepare Pre Amplification Mix

 

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

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


 

 

SECTION: cDNA Bead Purification
55. Mix sample with 40 µL Clean Up Beads (22% PEG) for a ratio of 1:0.8

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


SECTION: cDNA Bead Purification
57. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Bead Purification
58. Wash with 100 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: cDNA Bead Purification
59. Repeat wash step once more
SECTION: cDNA Bead Purification
60. Air dry beads for 5 min

 

SECTION: cDNA Bead Purification
61. Elute cDNA in 10 µL UltraPure Water

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

SECTION: cDNA Quantification and Quality Check
63. Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
SECTION: cDNA Quantification and Quality Check
64. Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits. 

 

 

SECTION: Library Preparation
65. 
 

SECTION: Library Preparation
66. Prepare Fragmentation Mix

 

 

 

SECTION: Library Preparation
67. Vortex the Fragmentation Mix for5 s and immediately proceed to step 67

SECTION: Library Preparation
68. Incubate the Fragmentation reaction as follows:

 



 

SECTION: Adapter Ligation
69. Prepare Adapter Ligation Mix


 


SECTION: Adapter Ligation
70. Add 6.7 µL  Adapter Ligation Mix to each replicate
SECTION: Adapter Ligation
71. Incubate for 15 min at 20 °C

 

SECTION: Adapter Ligation
72. Add 37.3 µL Buffer EB to Samples

SECTION: Adapter Ligation
73. Mix Sample with 26 µL SPRI select beads
 

 

SECTION: Adapter Ligation
74. Incubate for 5 min at 20 °C
SECTION: Adapter Ligation
75. Place the plate on the magnet stand until clear and transfer76 µL supernatant to clean well.  
 


SECTION: Adapter Ligation
76. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Adapter Ligation
79. Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Adapter Ligation
80. Repeat wash step once more
SECTION: Adapter Ligation
81. Air dry beads for 5 min

 

SECTION: Adapter Ligation
82. Elute samples in 11 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water) for 5 min

SECTION: Sample Collection
6. Prepare Lysis Buffer according to the number of samples

 

SECTION: Sample Collection
7. Add300 µL of Lysis Buffer to a 2 mL tube
 

SECTION: Sample Collection
8. Weigh the tissue and add to tube
SECTION: Sample Collection
9. Homogenize the tissue, while making sure to keep the samples cold
 

SECTION: Sample Collection
10. Dilute the homogenized lysate with additional Lysis Buffer for a total of 100 µL. Ideally, there should be roughly 100-500 ng of RNA in each sample.
 


SECTION: RNA Extraction
11. Add 135 µL Isopropanol



SECTION: RNA Extraction
12. Shake for 5 min at 20 °C at 1400 RPM
SECTION: RNA Extraction
13. Add 70 µL Cleanup Beads (22% PEG)



SECTION: RNA Extraction
14. Shake for 5 min at 20 °C at 1400 RPM




SECTION: RNA Extraction
15. Place on magnet stand until clear (~3 min) and then discard supernatant
SECTION: RNA Extraction
16. Add 300 µL Isopropanol



SECTION: RNA Extraction
17. Shake for 2 min at 20 °C at 1400 RPM
SECTION: RNA Extraction
18. Place on magnet stand until clear (~3 min) and then discard supernatant
SECTION: RNA Extraction
19. Wash with 300 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant

SECTION: RNA Extraction
20. Repeat wash step once more


 

SECTION: RNA Extraction
21. Air dry beads for 3 min

 

SECTION: RNA Extraction
22. Elute Extracted RNA by adding  20 µL H2O and resuspending the beads
SECTION: RNA Extraction
23. Shake for 5 min at 20 °C at 1400 RPM

SECTION: DNase I Digestion
24. Transfer 8 µL of Extracted RNA to 96-well plate

SECTION: DNase I Digestion
25. Prepare DNase I Mix 

 

 
 
SECTION: DNase I Digestion
26. Add 2 µL of DNase I Mix to each well



SECTION: DNase I Digestion
27. Incubate DNase I Mix and beads for 10 min at 20 °C


SECTION: DNase I Digestion
28. Heat inactivate the DNase I by adding 1 µL of EDTA (100 mM) and incubating for 5 min at 65 °C



SECTION: DNase I Digestion
29. Place plate on magnet stand until clear (~3 min) and discard the supernatant
SECTION: DNase I Digestion
30. Wash with 100 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: DNase I Digestion
31. Repeat wash step once more
SECTION: DNase I Digestion
32. Air dry beads for 5 min

 



SECTION: DNase I Digestion
33. Elute RNA with  15 µL H2O
SECTION: DNase I Digestion
34. Incubate for 5 min  at RT and transfer to a new PCR tube or plate

 

SECTION: RNA Quantification
35. Quantify the cDNA using the Quantifluor RNA System or equivalent following the manufacturer's protocol. Use 1 µL of RNA for quantification.

SECTION: RNA Quantification
36. Normalize the samples so they are all the same concentration. 
 

SECTION: RNA Quantification
37. Transfer 4 µL of the normalized RNA to a new plate. Return the unnormalized RNA to the freezer. 

SECTION: Reverse Transcription
38. Prepare Reverse Transcription Mix

 


 
SECTION: Reverse Transcription
39. Add 5 µL Reverse Transcription Mix to samples

SECTION: Reverse Transcription
40. Add 1 µL of Barcoded oligodT (E3V7NEXT) (10 µM) per well

SECTION: Reverse Transcription
41. Incubate for 90 min at 42 °C


SECTION: cDNA Pooling &amp; Purification
43. Pool all wells of one plate into a 2 mL tube

SECTION: cDNA Pooling &amp; Purification
42. Add 10 µL of Cleanup Beads (22% PEG) for each sample for a 1:1 ratio (e.g. 240 µL for 24 samples)

 

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


SECTION: cDNA Pooling &amp; Purification
45. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Pooling &amp; Purification
46. Wash with 1 mL of 80% EtOH while the tube is on the magnet, discard the supernatant

 

SECTION: cDNA Pooling &amp; Purification
47. Repeat wash step once more
SECTION: cDNA Pooling &amp; Purification
48. Air dry beads for 5 min

 

SECTION: cDNA Pooling &amp; Purification
49. Elute the beads in 17 µL of UltraPure Water 
SECTION: cDNA Pooling &amp; Purification
50. Incubate for 5 min  at RT and transfer to a new PCR tube or plate

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

 

SECTION: Exonuclease I Treatment
52. Mix each sample (20 µL per well) with 16 µL of Cleanup Beads (22% PEG) for a 1:0.8 ratio
SECTION: Exonuclease I Treatment
53. Incubate for 5 min at 20 Room temperature to allow binding of the cDNA onto beads


SECTION: Exonuclease I Treatment
54. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: Exonuclease I Treatment
55. Wash with 50 µL of 80% EtOH while the tube is on the magnet. Discard the supernatant
SECTION: Exonuclease I Treatment
56. Repeat wash step once more
SECTION: Exonuclease I Treatment
57. Air dry beads for 5 min

 

SECTION: Exonuclease I Treatment
58. Elute the beads in 20 µL of UltraPure Water 

SECTION: Exonuclease I Treatment
59. Incubate for 5 min  at RT and transfer to a new PCR tube or plate
SECTION: Full length cDNA Amplification
60. Prepare Pre Amplification Mix

 

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

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


 

 

SECTION: cDNA Bead Purification
63. Mix sample with 40 µL Clean Up Beads (22% PEG) for a ratio of 1:0.8

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


SECTION: cDNA Bead Purification
65. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Bead Purification
66. Wash with 100 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: cDNA Bead Purification
67. Repeat wash step once more
SECTION: cDNA Bead Purification
68. Air dry beads for 5 min

 

SECTION: cDNA Bead Purification
69. Elute cDNA in 10 µL UltraPure Water

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

SECTION: cDNA Quantification and Quality Check
71. Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
SECTION: cDNA Quantification and Quality Check
72. 

Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits. 

 


 

SECTION: Library Preparation
73. 
 

SECTION: Library Preparation
74. Prepare Fragmentation Mix

 

 

 

SECTION: Library Preparation
75. Vortex the Fragmentation Mix for5 s and immediately proceed to step 70

SECTION: Library Preparation
76. Incubate the Fragmentation reaction as follows:

 

 

SECTION: Adapter Ligation
77. Prepare Adapter Ligation Mix


 


SECTION: Adapter Ligation
78. Add 6.7 µL  Adapter Ligation Mix to each replicate
SECTION: Adapter Ligation
79. Incubate for 15 min at 20 °C

 

SECTION: Adapter Ligation
80. Add 37.3 µL Buffer EB to Samples

SECTION: Adapter Ligation
81. Mix Sample with 26 µL SPRI select beads
 

 

SECTION: Adapter Ligation
82. Incubate for 5 min at 20 °C
SECTION: Adapter Ligation
83. Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well
 


SECTION: Adapter Ligation
84. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Adapter Ligation
87. Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Adapter Ligation
88. Repeat wash step once more
SECTION: Adapter Ligation
89. Air dry beads for 5 min

 

SECTION: Adapter Ligation
90. Elute samples in 11 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water) for 5 min

SECTION: Library PCR
91. Transfer 10.5 µL of samples to clean wells

SECTION: Library PCR
92. Add 1 µL of Index Primer (Nextera i7, 5 uM) to each well

 

SECTION: Library PCR
94. Prepare Library PCR Mix by adding 12.5 µL 

 

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

 


 



SECTION: Library Double Size Selection
96. Add 25 µL Buffer EB to Index PCR

SECTION: Library Double Size Selection
97. Mix Index PCR with 26 µL SPRI select beads
 

 

SECTION: Library Double Size Selection
98. Incubate for 5 min at 20 °C
SECTION: Library Double Size Selection
99. Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well.  
 


SECTION: Library Double Size Selection
100. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Library Double Size Selection
103. Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Library Double Size Selection
104. Repeat wash step once more
SECTION: Library Double Size Selection
105. Air dry beads for 5 min

 

SECTION: Library Double Size Selection
106. Elute in 15 µL UltraPure Water. 
SECTION: Library Double Size Selection
107.  Incubate for 5 min and then place on magnet until clear. Transfer eluted library to new well. 
 

SECTION: Library QC
108. Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.

 


 

SECTION: Sequencing
109. Samples should be submitted according to your Sequencing Facility specifications. prime-seq is compatible with Illumina Sequencing.


At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Dual index sequencing can be done when using patterned flowcells. 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: Sample Preparation
6. Normalize the samples so they are all the same concentration. 
 

SECTION: Sample Preparation
7. Transfer 4 µL of the normalized RNA to a new plate. Return the unnormalized RNA to the freezer. 

SECTION: Reverse Transcription
8. Prepare Reverse Transcription Mix

 


 
SECTION: Reverse Transcription
9. Add 4 µL H2O
 

SECTION: Reverse Transcription
10. Add 5 µL Reverse Transcription Mix

SECTION: Reverse Transcription
11. Add 1 µL of Barcoded oligodT (E3V7NEXT) (10 µM) per well

SECTION: Reverse Transcription
12. Incubate for 90 min at 42 °C


SECTION: cDNA Pooling &amp; Purification
14. Pool all wells of one plate into a 2 mL tube

SECTION: cDNA Pooling &amp; Purification
13. Add 10 µL of Cleanup Beads (22% PEG) for each sample for a 1:1 ratio (e.g. 240 µL for 24 samples)

 

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


SECTION: cDNA Pooling &amp; Purification
16. Wash with 1 mL of 80% EtOH while the tube is on the magnet. Discard the supernatant

 

SECTION: cDNA Pooling &amp; Purification
17. Repeat wash step once more
SECTION: cDNA Pooling &amp; Purification
18. Air dry beads for 5 min

 

SECTION: cDNA Pooling &amp; Purification
19. Elute the beads in 17 µL of UltraPure Water 

SECTION: cDNA Pooling &amp; Purification
20. Incubate for 5 min  at RT and transfer to a new PCR tube or plate

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

 

SECTION: Exonuclease I Treatment
22. Mix each sample (20 µL per well) with 16 µL of Cleanup Beads (22% PEG) for a 1:0.8 ratio
SECTION: Exonuclease I Treatment
23. Incubate for 5 min at 20 Room temperature to allow binding of the cDNA onto beads


SECTION: Exonuclease I Treatment
24. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: Exonuclease I Treatment
25. Wash with 50 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: Exonuclease I Treatment
26. Repeat wash step once more
SECTION: Exonuclease I Treatment
27. Air dry beads for 5 min

 

SECTION: Exonuclease I Treatment
28. Elute the beads in 20 µL of UltraPure Water 

SECTION: Exonuclease I Treatment
29. Incubate for 5 min  at RT and transfer to a new PCR tube or plate
SECTION: Full length cDNA Amplification
30. Prepare Pre Amplification Mix

 

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

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


 

 

SECTION: cDNA Bead Purification
33. Mix sample with 40 µL Clean Up Beads (22% PEG) for a ratio of 1:0.8

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


SECTION: cDNA Bead Purification
35. Place the tube on the magnet stand until clear (~3 min) and discard supernatant
SECTION: cDNA Bead Purification
36. Wash with 100 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
SECTION: cDNA Bead Purification
37. Repeat wash step once more
SECTION: cDNA Bead Purification
38. Air dry beads for 5 min

 

SECTION: cDNA Bead Purification
39. Elute cDNA in 10 µL UltraPure Water

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

SECTION: cDNA Quantification and Quality Check
41. Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
SECTION: cDNA Quantification and Quality Check
42. Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits. 

 

 

SECTION: Library Preparation
43. 
 

SECTION: Library Preparation
44. Prepare Fragmentation Mix

 

 

 

SECTION: Library Preparation
45. Vortex the Fragmentation Mix for5 s and immediately proceed to step 70

SECTION: Library Preparation
46. Incubate the Fragmentation reaction as follows:

 



 

SECTION: Adapter Ligation
47. Prepare Adapter Ligation Mix


 


SECTION: Adapter Ligation
48. Add 6.7 µL  Adapter Ligation Mix to each replicate
SECTION: Adapter Ligation
49. Incubate for 15 min at 20 °C

 

SECTION: Adapter Ligation
50. Add 37.3 µL Buffer EB to Samples

SECTION: Adapter Ligation
51. Mix Samples with 26 µL SPRI select beads
 

 


SECTION: Adapter Ligation
52. Incubate for 5 min at 20 °C
SECTION: Adapter Ligation
53. Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well.  
 


SECTION: Adapter Ligation
54. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Adapter Ligation
57. Wash with 150 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant
SECTION: Adapter Ligation
58. Repeat wash step once more
SECTION: Adapter Ligation
59. Air dry beads for 5 min

 

SECTION: Adapter Ligation
60. Elute samples in 11 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water) for 5 min

SECTION: Library PCR
61. Transfer 10.5 µL of sample to clean wells

SECTION: Preparation
4. When running the protocol for the first time prepare Cleanup Beads (see end of the protocol)!

SECTION: Prepare Cleanup Beads
110. Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube

 

 


SECTION: Prepare Cleanup Beads
111. Incubate at 40 °C and vortex regularly until PEG is completely dissolved
SECTION: Prepare Cleanup Beads
112. Resuspend Sera-Mag Speed Beads carefully and pipette 1000 µL of bead suspension into a 1.5 mL tube
SECTION: Prepare Cleanup Beads
113. Place on magnet stand and remove storage buffer

SECTION: Prepare Cleanup Beads
114. Add 1000 µL of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads



SECTION: Prepare Cleanup Beads
115. Place on magnet stand and remove supernatant 
SECTION: Prepare Cleanup Beads
116. Repeat wash step one more time 
SECTION: Prepare Cleanup Beads
117. Add 900 µL TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads

SECTION: Prepare Cleanup Beads
118. Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
 

SECTION: Prepare Cleanup Beads
80. Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube

 

 


SECTION: Prepare Cleanup Beads
81. Incubate at 40 °C and vortex regularly until PEG is completely dissolved
SECTION: Prepare Cleanup Beads
82. Resuspend Sera-Mag Speed Beads carefully and pipette 1000 µL of bead suspension into a 1.5 mL tube
SECTION: Prepare Cleanup Beads
83. Place on magnet stand and remove storage buffer

SECTION: Prepare Cleanup Beads
84. Add 1000 µL of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads



SECTION: Prepare Cleanup Beads
85. Place on magnet stand and remove supernatant 
SECTION: Prepare Cleanup Beads
86. Repeat wash step one more time 
SECTION: Prepare Cleanup Beads
87. Add 900 µL TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads

SECTION: Prepare Cleanup Beads
88. Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
 

SECTION: Library PCR
93. Add 1 µL of Index Primer (TruSeq i5, 5 uM) to each well

 

SECTION: Library PCR
83. Transfer 10.5 µL of samples to clean wells

SECTION: Library PCR
84. Add 1 µL of Index Primer (Nextera i7, 5 uM) to each well

 

SECTION: Library PCR
85. Add 1 µL of Index Primer (TruSeq i5, 5 uM) to each well

 

SECTION: Library PCR
86. Prepare Library PCR Mix by adding 12.5 µL 

 

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

 


 



SECTION: Double Size Selection
88. Add 25 µL Buffer EB to Index PCR

SECTION: Double Size Selection
89. Mix Index PCR with 26 µL SPRI select beads
 

 

SECTION: Double Size Selection
90. Incubate for 5 min at 20 °C
SECTION: Double Size Selection
91. Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well.  
 


SECTION: Double Size Selection
92. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Double Size Selection
95. Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Double Size Selection
96. Repeat wash step once more
SECTION: Double Size Selection
97. Air dry beads for 5 min

 

SECTION: Double Size Selection
98. Elute in 15 µL UltraPure Water. 
SECTION: Double Size Selection
99.  Incubate for 5 min and then place on magnet until clear. Transfer eluted library to new well. 
 

SECTION: QC and quantification
100. Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.

 


 

SECTION: Sequencing
101. Samples should be submitted according to your Sequencing Facility specifications. prime-seq is compatible with Illumina Sequencing.


At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Dual index sequencing can be done when using patterned flowcells. 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: Library PCR
62. Add 1 µL of Index Primer (Nextera i7, 5 micromolar (µM)) to each well

 

SECTION: Library PCR
63. Add 1 µL of Index Primer (TruSeq i5, 5 micromolar (µM)) to each well

 

SECTION: Library PCR
64. Prepare Library PCR Mix by adding 12.5 µL 

 

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

 


 



SECTION: Library Double Size Selection
66. Add 25 µL Buffer EB to Index PCR

SECTION: Library Double Size Selection
67. Mix Index PCR with 26 µL SPRI select beads
 

 

SECTION: Library Double Size Selection
68. Incubate for 5 min at 20 °C
SECTION: Library Double Size Selection
69. Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well.  
 


SECTION: Library Double Size Selection
70. Mix supernatant with 10 µL SPRI select beads
 

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


SECTION: Library Double Size Selection
73. Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
SECTION: Library Double Size Selection
74. Repeat wash step once more
SECTION: Library Double Size Selection
75. Air dry beads for 5 min

 

SECTION: Library Double Size Selection
76. Elute in 15 µL UltraPure Water. 
SECTION: Library Double Size Selection
77.  Incubate for 5 min and then place on magnet until clear. Transfer eluted library to new well. 
 

SECTION: Library QC and quantification
78. Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.

 


 

SECTION: Sequencing
79. Samples should be submitted according to your Sequencing Facility specifications. prime-seq is compatible with Illumina Sequencing.


At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Dual index sequencing can be done when using patterned flowcells. 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: First Time Setup
6. When running the direct lysis protocol for the first time, prepare Bead Binding Buffer (see end of the protocol)!