TITLE: NEBNext Single Cell/ Low Input RNA Library Prep Kit for Illumina E6420 Protocol for Cells
AUTHORS: New England   Biolabs

[DESCRIPTION]
The NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® uses a template switching method to generate full length cDNAs directly from single cells or 2 pg – 200 ng RNA, followed by conversion to sequence-ready libraries using the Ultra™ II FS workflow. This unique workflow enables generation of the highest yields from a broad range of inputs, and superior transcript detection, while providing reliably consistent performance.

[BEFORE_START]
Please review the important information under the "Guidelines" & "Warnings” tabs before beginning.

Sample Recommendations 
This protocol is intended for isolated cultured or primary cells, but is not compatible with fixed cells. Cells should be intact and sorted in cell lysis buffer provided in the kit. See Section "Cell Collection and Lysis" for cell lysis buffer dilution and recommended volumes before use. Cells should be washed and resuspended in PBS prior to isolation/sorting. Carryover of media may affect the cDNA synthesis efficiency. 

Starting Material 
Isolated single, tens or hundred cells. 

Typical Yield of cDNA from a Reaction 
Actual yields will depend on the quality and quantity of the cell and the mRNA content of the sample. Typical cDNA yields range between 5–15 ng based on the PCR cycle recommendations provided in Section "cDNA Amplification by PCR". 

Typical Yield of Illumina Library from a Reaction 
Actual yields will depend on the quality and quantity of the input cDNA. Typical library yields range between 100 ng–1 µg based on the PCR cycle recommendations provided in Section "PCR Enrichment of Adaptor-ligated DNA". 

Notes
Keep all buffers and enzymes on ice, unless otherwise indicated

[GUIDELINES]
Overview 
The NEBNext Single Cell Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of total RNA inputs or RNA from cultured and primary cells into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user- friendly workflow also has minimal hands-on time. 
Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction of indexed libraries made from single cells and commercially available RNA and sequenced on an Illumina sequencing platform. 
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information. 

Workflow: Library Preparation for Illumina
  

Oligo Sequences
 


[STEPS]
SECTION: Sample and Reagents Preparation
1. Briefly centrifuge the tubes containing NEBNext Single Cell RT Enzyme Mix and Murine RNase Inhibitor to collect solutions to the bottom of the tubes, then place on ice.

SECTION: Sample and Reagents Preparation
2. Thaw all other frozen components at Room temperature (if the 10X NEBNext Cell Lysis Buffer appears cloudy after thawing, incubate briefly at 37 °C to clear up the solution).
SECTION: Sample and Reagents Preparation
3. Mix each component thoroughly, centrifuge briefly to collect solutions to the bottom of the tube, and then place on ice. Leave the 10X NEBNext Cell Lysis Buffer at Room temperature.
SECTION: Cell Collection and Lysis
4. If the carryover volume from cell isolation/sorting is < 1 µl, cells can be dispensed directly into 1X NEBNext Cell Lysis Buffer (without accounting for added volume). If carryover volume from cell isolation/sorting is ≥ 1 µl, skip to Step 8. 

Prepare 1X NEBNext Cell Lysis Buffer in an RNase-free tube as follows:

 



SECTION: Cell Collection and Lysis
5. Mix solution thoroughly by pipetting, avoiding bubbles. Centrifuge briefly to collect solution to the bottom of the tube. 
SECTION: Cell Collection and Lysis
6. Dispense cells directly into 5 µL. After dispensing, cells can be flash-frozen and stored at -80 °C for future use, or lysed as outlined in the next step. 

SECTION: Cell Collection and Lysis
7. Incubate at -80 Room temperature for 5 min and then proceed immediately to the next section ("Primer Annealing for First Strand Synthesis").

SECTION: Cell Collection and Lysis
8. If the carryover volume from cell isolation/sorting is ≥ 1 µl or the cells have already been collected in a solution with a volume ≥ 1 µl, prepare a Cell Lysis Buffer according to the table below, accounting for the carryover cell volume. Cells can be flash frozen and stored at -80 °C for future use or lysed as outlined in the next step.

 


SECTION: Cell Collection and Lysis
9. Incubate at -80 Room temperature for 5 min and then proceed immediately to the next section ("Primer Annealing for First Strand Synthesis").

SECTION: Primer Annealing for First Strand Synthesis
10. For carryover volumes < 1 µl or ≥ 1 µl, follow the corresponding protocol:
SECTION: Primer Annealing for First Strand Synthesis
11. Mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect solution to the bottom of the tube.
SECTION: Primer Annealing for First Strand Synthesis
12. Incubate for 5 min at 70 °C in a thermal cycler with the heated lid set to 105 °C, then hold at 4 °C until next step. 

During the above annealing step, prepare the components for the following step.
SECTION: Reverse Transcription (RT) and Template Switching
13. Vortex the NEBNext Single Cell RT Buffer, then prepare the RT mix in a separate tube as follows (adding NEBNext Single Cell RT Enzyme Mix last) on ice. 
 

 
 



SECTION: Reverse Transcription (RT) and Template Switching
14. Mix thoroughly by pipetting up and down several times, then centrifuge briefly to collect solutions to the bottom of tubes.
SECTION: Reverse Transcription (RT) and Template Switching
15. Combine 11 µL (above) with 9 µL (Step 12). Mix well by pipetting up and down at least 10 times, and centrifuge briefly. 
SECTION: Reverse Transcription (RT) and Template Switching
16. Incubate the reaction in a thermal cycler with the following steps and the heated lid set to 105 °C: 
90 min at 42 °C 
10 min at 70 °C 
Hold at 4 °C
 

SECTION: cDNA Amplification by PCR
17. Prepare cDNA amplification mix as follows:

 

SECTION: cDNA Amplification by PCR
18. Add 80 µL to 20 µL. Mix by pipetting up and down at least 10 times. 
SECTION: cDNA Amplification by PCR
19. Incubate the reaction in a thermal cycler with the following PCR cycling conditions and the heated lid set to 105 °C:

 


 

 

SECTION: Cleanup of Amplified cDNA
20. Allow the NEBNext Bead Reconstitution Buffer and the SPRI® beads (if stored at 4 °C) to warm to Room temperature for at least 30 min before use. Vortex SPRI Beads to resuspend well and prepare fresh 80 %.
SECTION: Cleanup of Amplified cDNA
21. Add 60 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for  3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Cleanup of Amplified cDNA
22. Incubate samples on the bench top for at least 5 min at Room temperature.

SECTION: Cleanup of Amplified cDNA
23. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Amplified cDNA
24. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
 

SECTION: Cleanup of Amplified cDNA
25. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

SECTION: Cleanup of Amplified cDNA
26. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Amplified cDNA
27. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Amplified cDNA
28. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 50 µL (dilute 1 X 1:10 in water).
SECTION: Cleanup of Amplified cDNA
29. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells.

SECTION: Cleanup of Amplified cDNA
30. Add 45 µL (4 Room temperature) to the eluted cDNA + bead mixture from the previous step for a second sample clean up. Mix well by pipetting up and down at least 10 times.
 


SECTION: Cleanup of Amplified cDNA
31. Incubate samples on the bench top for at least 5 min at Room temperature.

SECTION: Cleanup of Amplified cDNA
32. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Amplified cDNA
33. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
 

SECTION: Cleanup of Amplified cDNA
34. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
SECTION: Cleanup of Amplified cDNA
35. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Amplified cDNA
36. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Amplified cDNA
37. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 33 µL (provided in kit).
SECTION: Cleanup of Amplified cDNA
38. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
SECTION: Cleanup of Amplified cDNA
39. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube. 
 

SECTION: Assess Amplified cDNA Quality and Quantity on a Bioanalyzer
40. Run 1 µL from the previous step on a DNA High Sensitivity Chip.
 

 

 

SECTION: Fragmentation/End Prep
41. Ensure that the NEBNext Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use. 
SECTION: Fragmentation/End Prep
42. Vortex the NEBNext Ultra II FS Enzyme Mix 5–8 seconds prior to use and place on ice. 
 

SECTION: Fragmentation/End Prep
43. Add the following components to a 0.2 ml thin wall PCR tube on ice: 

 

SECTION: Fragmentation/End Prep
44. Vortex the reaction for 5 s and briefly spin in a microcentrifuge. 

SECTION: Fragmentation/End Prep
45. In a thermal cycler, with the heated lid set to 75 °C, run the following program: 
25 min at 37 °C 
30 min at 65 °C 
Hold at 4 °C
 

SECTION: Adaptor Ligation
46. Dilute (red) NEBNext Adaptor for Illumina by 25-fold (0.6 micromolar (µM)) in the NEBNext Adaptor Dilution Buffer (provided).
SECTION: Adaptor Ligation
47. Mix the NEBNext Ultra II Ligation Master Mix by pipetting up and down several times.
SECTION: Adaptor Ligation
48. Add the following components directly to the FS Reaction Mixture on ice:

 


 

SECTION: Adaptor Ligation
49. Set a 100 μl or 200 μl pipette to 50 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
 

SECTION: Adaptor Ligation
50. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off. 

SECTION: Adaptor Ligation
51. Add 3 µL to the ligation mixture from the previous step. 
 

SECTION: Adaptor Ligation
52. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C. 
 

SECTION: Cleanup of Adaptor-ligated DNA
53. 
 

SECTION: Cleanup of Adaptor-ligated DNA
54. If stored at 4 °C allow the SPRI beads to warm to 20 Room temperature for at least 30 min before use. Vortex SPRI beads to resuspend well and prepare fresh 80 %.

SECTION: Cleanup of Adaptor-ligated DNA
55. Add 57 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Cleanup of Adaptor-ligated DNA
56. Incubate samples on the bench top for at least 5 min at 20 Room temperature. 
SECTION: Cleanup of Adaptor-ligated DNA
57. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Adaptor-ligated DNA
58. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 

SECTION: Cleanup of Adaptor-ligated DNA
59. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of Adaptor-ligated DNA
60. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Adaptor-ligated DNA
61. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Adaptor-ligated DNA
62. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding  17 µL (dilute 1 X 1:10 in water). 
SECTION: Cleanup of Adaptor-ligated DNA
63. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 20 Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Cleanup of Adaptor-ligated DNA
64. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 15 µL to a new PCR tube. 

SECTION: Cleanup of Adaptor-ligated DNA
65. Proceed to PCR Enrichment of Adaptor-ligated DNA in the next section. 
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
66. 
 

Option A (Forward and Reverse Primers Supplied Separately)
Combine the following components in a sterile tube and then proceed to the next step: 

 



Option B (Forward and Reverse Primers Already Combined)
Combine the following components in a sterile tube and then proceed to the next step: 


 


SECTION: PCR Enrichment of Adaptor-ligated DNA
67. Set a 100 µl or 200 μl pipette to 40 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
SECTION: PCR Enrichment of Adaptor-ligated DNA
68. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling conditions:

 

 

SECTION: Cleanup of PCR Reaction
69. If stored at 4 °C allow the SPRI beads to warm to 20 Room temperature for at least 30 min before use. Vortex SPRI beads to resuspend well and prepare fresh 80 %.

SECTION: Cleanup of PCR Reaction
70. Add 45 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of PCR Reaction
71. Incubate samples on the bench top for at least 5 min at 20 Room temperature. 
SECTION: Cleanup of PCR Reaction
72. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of PCR Reaction
73. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 

SECTION: Cleanup of PCR Reaction
74. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Cleanup of PCR Reaction
75. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of PCR Reaction
76. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of PCR Reaction
77. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding  33 µL (dilute 1 X 1:10 in water). 
SECTION: Cleanup of PCR Reaction
78. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 20 Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Cleanup of PCR Reaction
79. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube. Libraries can be stored at -20 °C.

SECTION: Assess Library Quality and Quantity on a Bioanalyzer
80. Dilute library (from previous step) 5-fold in 0.1 X (inputs ≤ 1 ng may not require dilution to run on a Bioanalyzer). 
SECTION: Assess Library Quality and Quantity on a Bioanalyzer
81. Run 1 µL on a DNA High Sensitivity Chip. 

SECTION: Assess Library Quality and Quantity on a Bioanalyzer
82. Check that the electropherogram shows a narrow distribution with a peak size of 300–350 bp. 
 

 

SECTION: Primer Annealing for First Strand Synthesis
11. Mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect solution to the bottom of the tube.
SECTION: Primer Annealing for First Strand Synthesis
12. Incubate for 5 min at 70 °C in a thermal cycler with the heated lid set to 105 °C, then hold at 4 °C until next step. 

During the above annealing step, prepare the components for the following step.
SECTION: Reverse Transcription (RT) and Template Switching
13. Vortex the NEBNext Single Cell RT Buffer, then prepare the RT mix in a separate tube as follows (adding NEBNext Single Cell RT Enzyme Mix last) on ice. 
 

 
 

SECTION: Reverse Transcription (RT) and Template Switching
14. Mix thoroughly by pipetting up and down several times, then centrifuge briefly to collect solutions to the bottom of tubes.
SECTION: Reverse Transcription (RT) and Template Switching
15. Combine 11 µL (above) with 9 µL (Step 12 - the incubation step at the end of the Primer Annealing for First Strand Synthesis section). Mix well by pipetting up and down at least 10 times, and centrifuge briefly. 

SECTION: Reverse Transcription (RT) and Template Switching
16. Incubate the reaction in a thermal cycler with the following steps and the heated lid set to 105 °C: 
90 min at 42 °C 
10 min at 70 °C
Hold at 4 °C
 

SECTION: cDNA Amplification by PCR
17. Prepare cDNA amplification mix as follows:

 

SECTION: cDNA Amplification by PCR
18. Add 80 µL to 20 µL. Mix by pipetting up and down at least 10 times. 
SECTION: cDNA Amplification by PCR
19. Incubate the reaction in a thermal cycler with the following PCR cycling conditions and the heated lid set to 105 °C:

 

 

 

SECTION: Cleanup of Amplified cDNA
20. Allow the NEBNext Bead Reconstitution Buffer and the SPRI® beads (if stored at 4 °C) to warm to Room temperature for at least 30 min before use. Vortex SPRI Beads to resuspend well and prepare fresh 80 %.
SECTION: Cleanup of Amplified cDNA
21. Add 60 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Cleanup of Amplified cDNA
22. Incubate samples on the bench top for at least 5 min at Room temperature.

SECTION: Cleanup of Amplified cDNA
23. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Amplified cDNA
24. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
 

SECTION: Cleanup of Amplified cDNA
25. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

SECTION: Cleanup of Amplified cDNA
26. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Amplified cDNA
27. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Amplified cDNA
28. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 50 µL (dilute 1 X 1:10 in water).
SECTION: Cleanup of Amplified cDNA
29. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells.
SECTION: Cleanup of Amplified cDNA
30. Add 45 µL (4 Room temperature) to the eluted cDNA + bead mixture from the previous step for a second sample clean up. Mix well by pipetting up and down at least 10 times.
 

SECTION: Cleanup of Amplified cDNA
31. Incubate samples on the bench top for at least 5 min at Room temperature.

SECTION: Cleanup of Amplified cDNA
32. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Amplified cDNA
33. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
 

SECTION: Cleanup of Amplified cDNA
34. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
SECTION: Cleanup of Amplified cDNA
35. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Amplified cDNA
36. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Amplified cDNA
37. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 33 µL (provided in kit).
SECTION: Cleanup of Amplified cDNA
38. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand..
SECTION: Cleanup of Amplified cDNA
39. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube. 
 

SECTION: Assess Amplified cDNA Quality and Quantity on a Bioanalyzer
40. Run 1 µL from the previous step on a DNA High Sensitivity Chip.
 

 

 

SECTION: Fragmentation/End Prep
41. Ensure that the NEBNext Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use. 

SECTION: Fragmentation/End Prep
42. Vortex the NEBNext Ultra II FS Enzyme Mix 5–8 seconds prior to use and place on ice. 
 

SECTION: Fragmentation/End Prep
43. Add the following components to a 0.2 ml thin wall PCR tube on ice: 

 

SECTION: Fragmentation/End Prep
44. Vortex the reaction for 5 s and briefly spin in a microcentrifuge. 

SECTION: Fragmentation/End Prep
45. In a thermal cycler, with the heated lid set to 75 °C, run the following program: 
25 min at 37 °C
30 min at 65 °C
Hold at 4 °C
 


SECTION: Adaptor Ligation
46. Dilute (red) NEBNext Adaptor for Illumina by 25-fold (0.6 micromolar (µM)) in the NEBNext Adaptor Dilution Buffer (provided).
SECTION: Adaptor Ligation
47. Mix the NEBNext Ultra II Ligation Master Mix by pipetting up and down several times.
SECTION: Adaptor Ligation
48. Add the following components directly to the FS Reaction Mixture on ice:

 

 

SECTION: Adaptor Ligation
49. Set a 100 μl or 200 μl pipette to 50 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
 

SECTION: Adaptor Ligation
50. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off. 

SECTION: Adaptor Ligation
51. Add 3 µL to the ligation mixture from the previous step. 
 

SECTION: Adaptor Ligation
52. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C. 
 

SECTION: Cleanup of Adaptor-ligated DNA
53. 
 

SECTION: Cleanup of Adaptor-ligated DNA
54. If stored at 4 °C allow the SPRI beads to warm to 20 Room temperature for at least 30 min before use. Vortex SPRI beads to resuspend well and prepare fresh 80 %.
SECTION: Cleanup of Adaptor-ligated DNA
55. Add 57 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of Adaptor-ligated DNA
56. Incubate samples on the bench top for at least 5 min at 20 Room temperature. 
SECTION: Cleanup of Adaptor-ligated DNA
57. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Adaptor-ligated DNA
58. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 

SECTION: Cleanup of Adaptor-ligated DNA
59. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of Adaptor-ligated DNA
60. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of Adaptor-ligated DNA
61. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Adaptor-ligated DNA
62. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL (dilute 1X TE Buffer 1:10 in water). 

SECTION: Cleanup of Adaptor-ligated DNA
63. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 20 Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
SECTION: Cleanup of Adaptor-ligated DNA
64. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 15 µL to a new PCR tube. 
SECTION: Cleanup of Adaptor-ligated DNA
65. Proceed to PCR Enrichment of Adaptor-ligated DNA in the next section. 
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
66. 
 

Option A (Forward and Reverse Primers Supplied Separately)
Combine the following components in a sterile tube and then proceed to the next step: 
 


Option B (Forward and Reverse Primers Already Combined)
Combine the following components in a sterile tube and then proceed to the next step: 
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
67. Set a 100 µl or 200 μl pipette to 40 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
SECTION: PCR Enrichment of Adaptor-ligated DNA
68. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling conditions:

 

 

SECTION: Cleanup of PCR Reaction
69. If stored at 4 °C allow the SPRI beads to warm to 20 Room temperature for at least 30 min before use. Vortex SPRI beads to resuspend well and prepare fresh 80 %.
SECTION: Cleanup of PCR Reaction
70. Add 45 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of PCR Reaction
71. Incubate samples on the bench top for at least 5 min at 20 Room temperature. 
SECTION: Cleanup of PCR Reaction
72. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of PCR Reaction
73. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 

SECTION: Cleanup of PCR Reaction
74. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of PCR Reaction
75. Repeat previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol.
SECTION: Cleanup of PCR Reaction
76. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of PCR Reaction
77. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding  33 µL (dilute 1 X 1:10 in water). 
SECTION: Cleanup of PCR Reaction
78. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 20 Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
SECTION: Cleanup of PCR Reaction
79. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube. Libraries can be stored at -20 °C.
SECTION: Assess Library Quality and Quantity on a Bioanalyzer
80. Dilute library (from previous step) 5-fold in 0.1X TE Buffer (inputs ≤ 1 ng may not require dilution to run on a Bioanalyzer). 
SECTION: Assess Library Quality and Quantity on a Bioanalyzer
81. Run 1 µL on a DNA High Sensitivity Chip. 

SECTION: Assess Library Quality and Quantity on a Bioanalyzer
82. Check that the electropherogram shows a narrow distribution with a peak size of 300–350 bp.