TITLE: NEBNext Single Cell/ Low Input RNA Library Prep Kit for Illumina Protocol for Low Input RNA E6420
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 to be used for total RNA. 
The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts), divalent cation chelating agents (e.g. EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol). If an excess amount of genomic DNA is present in RNA samples, an optional DNase I treatment could be peformed. Inactivate/remove DNase I after treatment. 
Assess quality of the input RNA by running input RNA on an Agilent Bioanalyzer to determine the RNA Integrity Number (RIN). 

Starting Material 
2 pg–200 ng poly(A) tail-containing total RNA (DNA free), RIN score ≥ 8.0. 

Typical Yield of cDNA from a Reaction 
Actual yields will depend on the quality and quantity of the input RNA, the mRNA content of the sample, and the method used to purify the RNA. Typical cDNA yields range between 5–15 ng (for the lower RNA inputs) 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 Low Input RNA 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 10 X at Room temperature.
SECTION: Sample and Reagents Preparation
4. Thaw total RNA on ice prior to starting the protocol.
SECTION: Primer Annealing for First Strand Synthesis
5. To anneal cDNA Primer with total RNA samples, prepare the reaction as follows (on ice):

 


SECTION: Primer Annealing for First Strand Synthesis
6. Mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect solution to the bottom of the tubes.
SECTION: Primer Annealing for First Strand Synthesis
7. 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
8. Vortex the NEBNext Single Cell RT Buffer briefly, then prepare the RT mix in a separate tube as follows (adding NEBNext Single Cell RT Enzyme Mix last). 
 

 


SECTION: Reverse Transcription (RT) and Template Switching
9. 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
10. Combine 11 µL (above) with 9 µL (Step 7). Mix well by pipetting up and down at least 10 times, and centrifuge briefly. 

SECTION: Reverse Transcription (RT) and Template Switching
11. Incubate the reaction mix 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
12. Prepare cDNA amplification mix as follows:

 


SECTION: cDNA Amplification by PCR
13. Add 80 µL to 20 µL from Step 11. Mix by pipetting up and down at least 10 times.

SECTION: cDNA Amplification by PCR
14. Incubate the reaction in a thermal cycler with the following PCR cycling conditions and the heated lid set to 105 °C:

 



Recommended Number of PCR Cycles

 

 

 

SECTION: Cleanup of Amplified cDNA
15. 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
16. 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
17. Incubate samples on the bench top for at least 5 min at Room temperature.
SECTION: Cleanup of Amplified cDNA
18. 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
19. 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
20. 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
21. 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
22. 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
23. 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
24. 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
25. Add 45 µL (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
26. Incubate samples on the bench top for at least 5 min at Room temperature.
SECTION: Cleanup of Amplified cDNA
27. 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
28. 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
29. 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
30. 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
31. 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
32. 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
33. 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
34. 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
35. Run 1 µL from the previous step on a DNA High Sensitivity Chip.
 

 

 

SECTION: Cleanup of Adaptor-ligated DNA
49. If stored at 4 °C allow the SPRI® beads 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 Adaptor-ligated DNA
50. 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
51. Incubate samples on the bench top for at least 5 min at Room temperature.
SECTION: Cleanup of Adaptor-ligated DNA
52. 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
53. 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
54. 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 DNA targets.

SECTION: Cleanup of Adaptor-ligated DNA
55. 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
56. 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
57. Remove the tube/plate from the magnetic stand. Elute the DNA from the beads by adding 17 µL (dilute 1 X 1:10 in water).
SECTION: Cleanup of Adaptor-ligated DNA
58. 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 Adaptor-ligated DNA
59. 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
60. 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: Fragmentation/End Prep
36. 
 
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
37. Vortex the NEBNext Ultra II FS Enzyme Mix 5–8 seconds prior to use and place on ice. 
 

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

 


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

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

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

 


 

SECTION: Adaptor Ligation
44. 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
45. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off. 
SECTION: Adaptor Ligation
46. Add 3 µL to the ligation mixture from the previous step. 
 

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

SECTION: Cleanup of Adaptor-ligated DNA
48. 
 

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

SECTION: PCR Enrichment of Adaptor-ligated DNA
62. 
 

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
63. 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
64. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling conditions:

 



 


SECTION: Cleanup of PCR Reaction
65. 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
66. 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. Vortexing for 3–5
seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of PCR Reaction
67. Incubate samples on the bench top for at least 5 min at 20 Room temperature. 

SECTION: Cleanup of PCR Reaction
68. 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
69. 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
70. 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
71. 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
72. 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 PCR Reaction
73. 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
74. 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
75. 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
76. 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
77. Run 1 µL on a DNA High Sensitivity Chip. 

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