NEBNext Single Cell/ Low Input RNA Library Prep Kit for Illumina Protocol for Low Input RNA E6420
New England Biolabs
Abstract
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.
Attachments
Steps
Sample and Reagents Preparation
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.
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).
Mix each component thoroughly, centrifuge briefly to collect solutions to the bottom of the tube, and then place On ice. Leave the 10X at Room temperature.
Thaw total RNA On ice prior to starting the protocol.
Primer Annealing for First Strand Synthesis
To anneal cDNA Primer with total RNA samples, prepare the reaction as follows (On ice):
| A | B | C |
|---|---|---|
| COMPONENT | < 5 ng RNA VOLUME (µl) PER RXN | ≥ 5 ng RNA VOLUME (µl) PER RXN |
| Total RNA | Up to 8 µl | Up to 7 µl |
| (lilac) NEBNext Single Cell RT Primer Mix | 1 µl | 2 µl |
| Nuclease-free Water | Variable | Variable |
| Total Volume | 9 µl | 9 µl |
Mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect solution to the bottom of the tubes.
Incubate for 0h 5m 0s 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.
Reverse Transcription (RT) and Template Switching
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).
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| (lilac) NEBNext Single Cell RT Buffer | 5 µl |
| (lilac) NEBNext Template Switching Oligo | 1 µl |
| (lilac) NEBNext Single Cell RT Enzyme Mix | 2 µl |
| Nuclease-free Water | 3 µl |
| Total Volume | 11 µl |
Mix thoroughly by pipetting up and down several times, then centrifuge briefly to collect solutions to the bottom of tubes.
Combine 11µL (above) with 9µL (Step 7). Mix well by pipetting up and down at least 10 times, and centrifuge briefly.
Incubate the reaction mix in a thermal cycler with the following steps and the heated lid set to 105°C:
1h 30m 0s at 42°C
0h 10m 0s at 70°C
Hold at 4°C
cDNA Amplification by PCR
Prepare cDNA amplification mix as follows:
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| (orange) NEBNext Single Cell cDNA PCR Master Mix | 50 µl |
| (orange) NEBNext Single Cell cDNA PCR Primer | 2 µl |
| (white) NEBNext Cell Lysis Buffer (10X) | 0.5 µl |
| Nuclease-free Water | 27.5 µl |
| Total Volume | 80 µl |
Add 80µL to 20µL from Step 11. Mix by pipetting up and down at least 10 times.
Incubate the reaction in a thermal cycler with the following PCR cycling conditions and the heated lid set to 105°C:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 98°C | 45 seconds | 1 |
| Denaturation | 98°C | 10 seconds | 7-21* (See 'Recommended Number of PCR Cycles' table below) |
| Annealing | 62°C | 15 seconds | |
| Extension | 72°C | 3 minutes | |
| Final Extension | 72°C | 5 minutes | 1 |
| Hold | 4°C | ∞ |
Recommended Number of PCR Cycles
| A | B |
|---|---|
| TOTAL RNA | RECOMMENDED NUMBER OF PCR CYCLES* |
| 2 pg | 20-21 |
| 10 pg | 17-18 |
| 100 pg | 14-15 |
| 1 ng | 10-11 |
| 10 ng | 8-9 |
| 100 ng/200 ng | 7-8 |
*Note: The amount of RNA in your sample should be used to determine the appropriate number of PCR cycles.
Cleanup of Amplified cDNA
Allow the NEBNext Bead Reconstitution Buffer and the SPRI® beads (if stored at 4°C) to warm to Room temperature for at least 0h 30m 0s before use. Vortex SPRI Beads to resuspend well and prepare fresh 80%.
Add 60µL 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.
Incubate samples on the bench top for at least 0h 5m 0s at Room temperature.
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.
After 0h 5m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
Add 200µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 50µL (dilute 1X 1:10 in water).
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells.
Add 45µL (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.
Incubate samples on the bench top for at least 0h 5m 0s at Room temperature.
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.
After 0h 5m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
Add 200µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.
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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 33µL (provided in kit).
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells.
Place the tube/plate on the magnetic stand. After 0h 5m 0s (or when the solution is clear), transfer 30µL to a new PCR tube.
Assess Amplified cDNA Quality and Quantity on a Bioanalyzer
Run 1µL from the previous step on a DNA High Sensitivity Chip.
Fragmentation/End Prep
On ice until use. Vortex the NEBNext Ultra II FS Enzyme Mix 5–8 seconds prior to use and place On ice.
Add the following components to a 0.2 ml thin wall PCR tube On ice:
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| cDNA (Step 34) | 26 µl |
| (yellow) NEBNext Ultra II FS Reaction Buffer | 7 µl |
| (yellow) NEBNext Ultra II FS Enzyme Mix | 2 µl |
| Total Volume | 35 µl |
Vortex the reaction for 0h 0m 5s and briefly spin in a microcentrifuge.
In a thermal cycler, with the heated lid set to 75°C
0h 25m 0s at 37°C
0h 30m 0s at 65°C
Hold at 4°C
Adaptor Ligation
Dilute (red) NEBNext Adaptor for Illumina by 25-fold (0.6micromolar (µM)) in the NEBNext Adaptor Dilution Buffer (provided).
Mix the NEBNext Ultra II Ligation Master Mix by pipetting up and down several times.
Add the following components directly to the FS Reaction Mixture On ice:
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| FS Reaction Mixture (Step 40) | 35 µl |
| (red) NEBNext Ultra II Ligation Master Mix | 30 µl |
| (red) NEBNext Ligation Enhancer | 1 µl |
| (red) NEBNext Adaptor for Illumina* (diluted 1:25) | 2.5 µl |
| Total Volume | 68.5 µl |
*The NEBNext adaptor is provided in the NEBNext Oligo kit. NEB has several Oligo kit options, which are supplied separately from the library prep kit.
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.
Incubate at 20°C for 0h 15m 0s in a thermal cycler with the heated lid off.
Add 3µL to the ligation mixture from the previous step.
Mix well and incubate at 37°C for 0h 15m 0s with the heated lid set to ≥ 47°C.
Cleanup of Adaptor-ligated DNA
If stored at 4°C allow the SPRI® beads to warm to Room temperature for at least 0h 30m 0s before use. Vortex SPRI Beads to resuspend well and prepare fresh 80%.
Add 57µL 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.
Incubate samples on the bench top for at least 0h 5m 0s at Room temperature.
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.
After 0h 5m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Add 200µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Remove the tube/plate from the magnetic stand. Elute the DNA from the beads by adding 17µL (dilute 1X 1:10 in water).
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s 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.
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.
Place the tube/plate on the magnetic stand. After 0h 5m 0s (or when the solution is clear), transfer 15µL to a new PCR tube.
Proceed to PCR Enrichment of Adaptor-ligated DNA in the next section.
PCR Enrichment of Adaptor-ligated DNA
Option A (Forward and Reverse Primers Supplied Separately)
Combine the following components in a sterile tube and then proceed to the next step:
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| Adaptor Ligated DNA Fragments (Step 60) | 15 µl |
| (blue) NEBNext Ultra II Q5 Master Mix | 25 µl |
| (blue) Index Primer/i7 Primer*,** | 5 µl |
| (blue) Universal PCR Primer/i5 Primer*, ** | 5 µl |
| Total Volume | 50 µl |
*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations. **Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample.
Option B (Forward and Reverse Primers Already Combined)
Combine the following components in a sterile tube and then proceed to the next step:
| A | B |
|---|---|
| COMPONENT | VOLUME (µl) PER REACTION |
| Adaptor Ligated DNA Fragments (Step 60) | 15 µl |
| (blue) NEBNext Ultra II Q5 Master Mix | 25 µl |
| Index Primer Mix * | 10 µl |
| Total Volume | 50 µl |
*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations
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.
Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling conditions:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 98°C | 30 seconds | 1 |
| Denaturation | 98°C | 10 seconds | 8* |
| Annealing/ Extension | 65°C | 75 seconds | |
| Final Extension | 65°C | 5 minutes | 1 |
| Hold | 4°C | ∞ |
- If your cDNA input is outside the input range of 1 ng–20 ng, adjust the PCR cycle numbers accordingly. We recommend a minimum of 3 PCR cycles for all of the original molecules to make it into the final library. For cDNA yield of 100 pg we recommend testing 12 PCR cycles. For cDNA input of 1 ng–20 ng, the typical Illumina library yield, using 8 PCR cycles, is 100 ng–1 μg.
| A | B |
|---|---|
| INPUT IN THE FRAGMENTATION/END PREP REACTION* | # CYCLES REQUIRED |
| 100 pg–1 ng | 9–12 |
| 1 ng–20 ng | 6–9 |
| 20 ng–100 ng | 3–6 |
- It is possible to normalize the cDNA input into the Fragmentation/End Prep Reaction so that all libraries start out with a similar amount of cDNA.
Cleanup of PCR Reaction
If stored at 4°C allow the SPRI beads to warm to 20Room temperature for at least 0h 30m 0s before use. Vortex SPRI beads to resuspend well and prepare fresh 80%.
Add 45µL 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.
Incubate samples on the bench top for at least 0h 5m 0s at 20Room temperature.
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.
After 0h 5m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Add 200µL to the tube/ plate while in the magnetic stand. Incubate at 20Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33µL (dilute 1X 1:10 in water).
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at 20Room 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.
Place the tube/plate on the magnetic stand. After 0h 5m 0s (or when the solution is clear), transfer 30µL to a new PCR tube. Libraries can be stored at -20°C.
Assess Library Quality and Quantity on a Bioanalyzer
Dilute library (from previous step) 5-fold in 0.1X (inputs ≤ 1 ng may not require dilution to run on a Bioanalyzer).
Run 1µL on a DNA High Sensitivity Chip.
Check that the electropherogram shows a narrow distribution with a peak size of 300–350 bp.
