TITLE: mcSCRB-seq2 protocol
AUTHORS: Lucas Esteban Wange, Johannes JWB Bagnoli, Aleksandar  Janjic, Wolfgang Enard

[DESCRIPTION]
mcSCRB-seq2 is the improved version of our published protocol molecular crowding SCRB-seq (mcSCRB-seq) (https://www.nature.com/articles/s41467-018-05347-6/). 

Following changes were introduced:
re-evaluted the Lysis Buffer formulation, making it more robust against RNAses
added additional dCTP in the Reverse Transcription leading to higher yield by enhanced Template switching
updated the oligo dT primers to be compatible with 10x's three prime gene expression layout (see prime-seq for more information)
changed to KapaHifi for PreAmplification as it produces less chimeric rerads compared to Tera Polymerase
increased Primer concentration in PreAmp (SingV6)
included an option for cheaper library prep with NEBNext 

[BEFORE_START]
Wipe bench surfaces with RNAse Away and keep working environment clean.

[GUIDELINES]
All reagents and plasticware 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 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
6. Before starting clean all surfaces and pipettes with RNase Away. 
Apply RNase Away for 3-5 minutes and wipe away with a clean tissue.
SECTION: Preparation
7. When running the protocol for the first time, prepare Cleanup Beads as described in Appendix 1.

 


SECTION: Preparation
8. When running the protocol for the first time, prepare Pooling Beads as described in Appendix 2.

 

SECTION: Reverse Transcription
10. Prepare Reverse Transcription Mix

 

 

SECTION: Reverse Transcription
13. Add 5 µL Reverse Transcription Mix to each well using a repeater pipette or a liquid handling robot like the Mantis.

SECTION: Reverse Transcription
14. Incubate for 90 min at 42 °C in a Thermal Cycler with heated lid 105 °C .

SECTION: cDNA Pooling & Purification
15. Mix each well (10 µL per well) with 10 µL of Pooling Beads  for a 1:1 ratio. Pooling Beads can be added using a repeater pipette.

 

SECTION: cDNA Pooling & Purification
16. Pool all wells of one plate into a 2 mL tube or 15 mL tube when pooling up to 384 cells.

 

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


SECTION: cDNA Pooling & Purification
18. Place the tube on the magnet stand until clear, approximately  5 min  and discard supernatant.

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

 

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

 

SECTION: cDNA Pooling & Purification
22. Resuspend the beads in 17 µL  of UltraPure Water and remove from Magnet.

SECTION: Exonuclease I Treatment
25. Incubate as follows in a Thermal Cycler with heated Lid 105 °C :

 


SECTION: Full length cDNA Amplification
35. Prepare Pre Amplification Mix. Adjust to the number of pools.

 

SECTION: Full length cDNA Amplification
36. Add 30 µL Pre Amplification Mix to each pool.

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


 

 

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

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


SECTION: cDNA Bead Purification
40. Place the plate on the magnet rack until clear (~5 min ) .

SECTION: cDNA Bead Purification
42. Wash with 100 µL of 80% EtOH while the plate is on the magnet.
SECTION: cDNA Bead Purification
44. Repeat wash step once more.  

SECTION: cDNA Bead Purification
45. Air dry beads for 5 min

 

SECTION: cDNA Quantification and Quality Check
48. Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
SECTION: cDNA Quantification and Quality Check
49. Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits. Other instruments like the Tape Station or the Fragment Analyzer can be used as well but make sure to not waste more than 4 µL of your cDNA.

 

 

SECTION: Exonuclease I Treatment
26. Mix sample with 20 µL Clean Up Beads (22% PEG) for a ratio of 1:1
 

SECTION: Exonuclease I Treatment
27. Incubate for 5 min at 20 °C


SECTION: Exonuclease I Treatment
28. Place the plate on the magnet stand until clear (~5 min  ) and discard supernatant.

SECTION: Exonuclease I Treatment
29. Wash with 100 µL of 80% EtOH while the plate is on the magnet.
SECTION: Exonuclease I Treatment
31. Repeat wash step once more.  

SECTION: Exonuclease I Treatment
32. Air dry beads for 5 min

 

50. 
 

SECTION: Library Preparation - Fragmentation
53. Prepare Fragmentation Mix
 

 

SECTION: Library Preparation - Fragmentation
55.  Vortex the Fragmentation Mix for 5 s  and immediately proceed to next step.

 

SECTION: Library Preparation - Fragmentation
52. Start by setting up the Thermo Cycler to be able to immediately proceed to the incubation after adding the Fragmentation Mix to the cDNA.
 


 

SECTION: Library Preparation - Adapter Ligation
57. Prepare Adapter Ligation Mix:

 

SECTION: Library Preparation - Adapter Ligation
58. Add 6.7 µL  Adapter Ligation Mix to each replicate.

SECTION: Library Preparation - Adapter Ligation
59. Incubate for 15 min  at  20 °C 

 


SECTION: Library Preparation - Double size selection
60. Add 37.3 µL  Buffer EB to Samples for a total of 50 µL 

SECTION: Library Preparation - Double size selection
61. Mix Index PCR with 25 µL SPRI select beads (ratio of 0.5x). 

 


SECTION: Library Preparation - Double size selection
62. Incubate for 5 min  at room temperature.

SECTION: Library Preparation - Double size selection
63. Place the plate on the magnet stand until clear and transfer 75 µL supernatant to a clean well.

 


SECTION: Library Preparation - Double size selection
64. Mix supernatant with 10 µL  SPRI select beads (ratio of 1:0.7)

SECTION: Library Preparation - Double size selection
65. Incubate for 5 min  at Room temperature 

SECTION: Library Preparation - Double size selection
66. Place the plate on the magnet stand until clear.
SECTION: Library Preparation - Double size selection
68. Wash with 150 µL  of 80% EtOH while the plate is on the magnet.

SECTION: Library Preparation - Double size selection
70. Repeat wash step once more.  

SECTION: Library Preparation - Double size selection
71. Air dry beads for 5 min  

 


SECTION: Library Preparation - Double size selection
72. Take plate off the magnet and resuspend samples in 10.5 µL  0.1X TE (dilute 1X TE Buffer 1:10 in water).


SECTION: Library Preparation - Double size selection
74. Place plate on magnet and transfer samples to clean wells.
SECTION: Library Preparation - Library PCR and Indexing
76. Add 1 µL  of Nextera i7 Index Primer (5 micromolar (µM) ) to each well.

SECTION: Library Preparation - Library PCR and Indexing
78. Add 12.5 µL of 2x Q5 Master Mix (NEBNext Ultra II) to each well.

 


SECTION: Library Preparation - Library PCR and Indexing
80. Adjust the number of cycles based on total cDNA input. 
As a general guide we recommend: 


 

SECTION: Library Preparation - Final Double Size Selection and Clean Up
81. Add 25 µL  Buffer EB to Index PCR.

SECTION: Library Preparation - Final Double Size Selection and Clean Up
82. Mix Index PCR with 25 µL  SPRI select beads (ratio of 1:0.5) 

SECTION: Library Preparation - Final Double Size Selection and Clean Up
83. Incubate for 5 min  at Room temperature 

SECTION: Library Preparation - Final Double Size Selection and Clean Up
84. Place the plate on the magnet stand until clear and transfer 75 µL supernatant to a clean well. 

 

SECTION: Library Preparation - Final Double Size Selection and Clean Up
85. Mix supernatant with 10 µL SPRI select beads (ratio of 1:0.7)

SECTION: Library Preparation - Final Double Size Selection and Clean Up
86. Incubate for 5 min  at Room temperature .
SECTION: Library Preparation - Final Double Size Selection and Clean Up
87. Place the plate on the magnet stand until clear.
SECTION: Library Preparation - Final Double Size Selection and Clean Up
89. Wash with 150 µL  of 80% EtOH while the plate is on the magnet. 

SECTION: Library Preparation - Final Double Size Selection and Clean Up
91. Repeat wash step once more.   

SECTION: Library Preparation - Final Double Size Selection and Clean Up
92. Air dry beads for 5 min .

 


SECTION: Library Preparation - Final Double Size Selection and Clean Up
93. Elute in 15 μl UltraPure Wate r. 107 Incubate for 00:05:00 and then place on magnet until clear. Transfer eluted library to new well. Stopping point. The libraries can be safely stored at -20 °C until they will be QCed and sequenced. Library QC 45m
SECTION: Appendix: Prepare Clean Up Beads (SPRI 22% PEG)
102. Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube

 

 


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

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



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

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

SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
111. Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube

 

 


SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
112. Incubate at 40 °C and vortex regularly until PEG is completely dissolved
SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
113. Resuspend Sera-Mag Speed Beads carefully and pipette 200 µL of bead suspension into a 1.5 mL tube
SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
114. Place on magnet stand and remove storage buffer

SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
115. Add 1000 µL of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads



SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
116. Place on magnet stand and remove supernatant 
SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
117. Repeat wash step one more time 
SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
118. Add 900 µL TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads

SECTION: Appendix: Prepare Pooling Beads (SPRI 22% PEG 5 times less Beads)
119. Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
 

SECTION: Sample Collection
1. Prepare Lysis Buffer for the number of plates you intend to use. 
  Ingredient1xNxGen RNAi (40U/ul)0.125Phusion HF0.008H2O3.867Total4
 
SECTION: Sample Collection
2. Distribute 4 µL  PPi Lysis Buffer per well using a repeater pipette (stepper) or similar.
  
 Add  1 µL  barcoded oligo-dT primer [2 μM] (E3V7) to each well using a multichannel pipette.

 


SECTION: Sample Collection
3. Deposit one cell per well in the Lysis plate using e.g. FACS
 

SECTION: Sample Collection
4. Immediately after sorting close plate tightly with an aluminum seal.
SECTION: Sample Collection
5. In a cooled centrifuge, spin down the plate  1000 x g, 30 s, 4 °C  and place immediately on dry ice.
Store plates at -70 °C  or lower for up to 6 month before processing.

SECTION: Reverse Transcription
12. Spin down in a pre-cooled centrifuge 1000 rcf, 30 s, 4 °C .

SECTION: Reverse Transcription
11. Gently thaw plates on ice for 1 min at most.

SECTION: Library Preparation
51. Use one fourth of your cDNA 2.5 µL but not more than 20 ngin total . If your cDNA concentration is higher than 8 ng/µL, dilute accordingly.
 

SECTION: cDNA Pooling & Purification
23. Incubate for 5 min  and place on magnet to transfer supernatant to a new well of a 96 well PCR plate.

SECTION: Exonuclease I Treatment
33. Remove the plate from the magnet and resuspend the beads in 20 µL  of UltraPure Water.

SECTION: Exonuclease I Treatment
34. Incubate for 5 min  and place on magnet to transfer supernatant to a new well.

SECTION: cDNA Bead Purification
46. Remove from Magnet and resuspend the beads in 10 µL  of UltraPure Water.

SECTION: cDNA Bead Purification
47. Incubate for 5 min  and place on magnet to transfer supernatant to a new well.


 


SECTION: Library Preparation - Library PCR and Indexing
77. Add 1 µL  of TruSeq i5 Index Primer (5 micromolar (µM) ) to each well.

SECTION: Library Preparation - Library PCR and Indexing
75. 
 

SECTION: Library Preparation - Library PCR and Indexing
79. Incubate Library PCR reaction as follows with the heated Lid set to 105 °C :

 

SECTION: Exonuclease I Treatment
24. Add 2 µL of Exonuclease I Buffer (10x) and 1 µL of Exonuclease I per pool. 

 

SECTION: Exonuclease I Treatment
30. Discard the supernatant and keep plate on the magnet.
SECTION: cDNA Bead Purification
41. Discard supernatant and keep plate on the magnet.
SECTION: cDNA Bead Purification
43. Discard supernatant and keep plate on the magnet.
SECTION: Reverse Transcription
9. 
 

SECTION: Library Preparation - Fragmentation
54. Add 2.5 µL of cDNA (between 0.5 & 8 ng/µl) to a new well of a 96 well plate and add 3.5 µL of Fragmentation Mix.



SECTION: Library Preparation - Fragmentation
56. Place plate containing the samples into Thermal Cycler and start incubation by skiping the initial 4 °C  hold.

SECTION: Library Preparation - Double size selection
67. Discard supernatant and keep plate on magnet.
SECTION: Library Preparation - Double size selection
69.  Discard the supernatant and keep plate on magnet.
SECTION: Library Preparation - Double size selection
73. Elute for 5 min minutes.
SECTION: Library Preparation - Final Double Size Selection and Clean Up
88. Discard supernatant and keep plate on the magnet.
SECTION: Library Preparation - Final Double Size Selection and Clean Up
90. Discard supernatant and keep plate on the magnet.
SECTION: Library Preparation - Final Double Size Selection and Clean Up
94. Take plate off the magnet and resuspend samples in 15 µL  UltraPure Water.


SECTION: Library Preparation - Final Double Size Selection and Clean Up
95. Elute for 5 min minutes.
SECTION: Library Preparation - Final Double Size Selection and Clean Up
96. Transfer 15 µL clean sequencing Library to a 0.5 mL tube for storage.

 


SECTION: Library Preparation - Quantification and QC
97. Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent following the manufacturer's protocol. Use 1 µL  of the final Library for quantification.

 


SECTION: Library Preparation - Quantification and QC
98. Quality control and Quantify the Library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits. Other instruments like the Tape Station or the Fragment Analyzer can be used as well.

 

 

SECTION: Sequencing
100. 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 Reads (i7+i5) and 28 cycles for the 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 are sufficient.

Some potential sequencing options:

 



SECTION: Library Preparation - Quantification and QC
99. Quantify the Library Molarity using the Bioanalyzer2000 (or else) software. Make sure to set the Region from 200 bp to 1000 bp. The Bioanalyzer will calculate the Molarity based on fragment size and Fluorescence intensity.
We usually aim for a Library concentration of 10nM (10000pM) to submit for sequencing. Lower or higher molarities might be required depending on the sequencing provider.
SECTION: Sequencing
101. Sequencing Depth should be adjusted to the scientific question, for example for broad cell type classification few read per cell between 10 k and 25 k are usually sufficient. For in depth transcriptome anaylsis between 100k - 500k reads per cell are adequate. Please note that these are just general remarks and library complexity may differ considerably depending on cell type, state and quality of the input.