Protocol for Cells: cDNA Synthesis and Amplification (NEB# E6421)

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	This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
	This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.
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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 1.2 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/input RNA and the mRNA content of the sample. Typical cDNA yields range between 5–15 ng based on the PCR cycle recommendations provided in Section 1.5.

Notes

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

1.1. Sample and Reagents Preparation

1.1.1. 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.

1.1.2. 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).

1.1.3. 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.

1.2.Cell Collection and Lysis

1.2.1. 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 1.2.5.

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

COMPONENT	VOLUME (μl) PER REACTION
(white) NEBNext Cell LysisBuffer (10X)	0.5 µl
(white) Murine RNase Inhibitor	0.25 µl
Nuclease-free water	4.25 µl
Total Volume	5 µl

1.2.2. Mix solution thoroughly by pipetting, avoiding bubbles. Centrifuge briefly to collect solution to the bottom of the tube.

1.2.3. Dispense cells directly into 5 µl 1X Cell Lysis Buffer. After dispensing, cells can be flash-frozen and stored at -80°C for future use, or lysed as outlined in Step 1.2.4.

1.2.4. Incubate at room temperature for 5 minutes and then proceed immediately to Section 1.3

1.2.5. 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 Step 1.2.6.

REAGENT	VOLUME (μl) PER REACTION
Carryover Cell Volume	1-5 µl
(white) NEBNext Cell LysisBuffer (10X)	0.8 µl
(white) Murine RNase Inhibitor	0.4 µl
Nuclease-free water	Variable (based on carryover cell volume)
Total Volume	8 µl

1.2.6.Incubate at room temperature for 5 minutes and then proceed immediately to Section 1.3.

1.3.Primer Annealing for First Strand Synthesis

Follow Step 1.3.1A for carryover volumes < 1 µl.

Follow Step 1.3.1B for carryover volumes ≥ 1 µl.

1.3.1. To anneal cDNA Primer with RNA templates in the sample, prepare the reaction as follows (on ice):

1.3.1A. Carryover Volume < 1 µl

COMPONENT	VOLUME (μl) PER REACTION
Lysed Cell (Step 1.2.4)	5 µl
(lilac) NEBNext Single Cell RT Primer Mix	1 µl
Nuclease-free water	3 µl
Total Volume	9 µl

Continue to step 1.3.2.

1.3.1B. Carryover Volume ≥ 1 µl

COMPONENT	VOLUME (μl) PER REACTION
Lysed Cell (Step 1.2.6)	8 µl
(lilac) NEBNext Single Cell RT Primer Mix	1 µl
Total Volume	9 µl

1.3.2. Mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect solution to the bottom of the tube.

1.3.3. Incubate for 5 minutes at 70°C in a thermocycler with the heated lid set to 105°C, then hold at 4ºC until next step.

During the above annealing step, prepare the components for the following step.

1.4.Reverse Transcription (RT) and Template Switching

1.4.1. 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.

Note: It is important to vortex the buffer prior to use for optimal performance.

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

1.4.2. Mix thoroughly by pipetting up and down several times, then centrifuge briefly to collect solutions to the bottom of tubes.

1.4.3. Combine 11 µl of the RT mix (above) with 9 µl of the annealed sample (Step 1.3.3). Mix well by pipetting up and down at least 10 times, and centrifuge briefly.

1.4.4.Incubate the reaction in a thermocycler with the following steps and the heated lid set to 105°C:

90 minutes at 42°C
10 minutes at 70°C
Hold at 4°C

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

1.5. cDNA Amplification by PCR

1.5.1. Prepare cDNA amplification mix as follows:

COMPONENT	VOLUME (μl) PER REACTION
(orange) NEBNext Single Cell cDNA PCR Master Mix	50 µl
(orange) NEBNext Single Cell cDNA PCR Primer	2 µl
Nuclease-free water	28 µl
Total Volume	80 µl

1.5.2. Add 80 µl cDNA amplification mix to 20 µl of the sample from Step 1.4.4. Mix by pipetting up and down at least 10 times.

1.5.3. Incubate the reaction in a thermocycler with the following PCR cycling conditions and the heated lid set to 105°C:

Recommended Number of PCR Cycles

*Note: The amount of RNA in your sample should be used to determine the appropriate number of PCR cycles. If you are starting with single cells not listed above, a PCR cycle titration can be done to determine the appropriate number of PCR cycles for your sample.

For the various inputs listed above, the recommended PCR cycles will typically result in cDNA yields between 5–15 ng. The total RNA used for the above recommendations is Universal Human Reference (UHR) RNA. When using other sources of starting material or a different cell type some optimization may be necessary due to variations in mRNA amounts.

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

1.6. Cleanup of Amplified cDNA

1.6.1. Allow the NEBNext Bead Reconstitution Buffer and the SPRI® beads (if stored at 4°C) to warm to room temperature for at least 30 minutes before use. Vortex SPRI Beads to resuspend well and prepare fresh 80% ethanol.

1.6.2. Add 60 µl (0.6X of sample volume) resuspended beads 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.

1.6.3. Incubate samples on the bench top for at least 5 minutes at room temperature.

1.6.4. 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.

1.6.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA (Caution: do not discard the beads).

1.6.6. Add 200 µl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

1.6.7. Repeat Step 1.6.6 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.

1.6.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of cDNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

1.6.9. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 50 µl of 0.1X TE (dilute 1X TE Buffer 1:10 in water).

1.6.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells.

1.6.11. Add 45 µl of (room temperature) NEBNext Bead Reconstitution Buffer to the eluted cDNA + bead mixture from Step 1.6.10 for a second sample clean up. Mix well by pipetting up and down at least 10 times (Caution: Skipping this additional cleanup step may reduce overall cDNA purity).

1.6.12. Incubate samples on the bench top for at least 5 minutes at room temperature.

1.6.13. 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.

1.6.14. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA (Caution: do not discard the beads).

1.6.15. Add 200 µl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

1.6.16. Repeat Step 1.6.15 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.

1.6.17. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of cDNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

1.6.18. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 33 µl of 1X TE (provided in kit).

1.6.19. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes 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.

1.6.20. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 µl to a new PCR tube.

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

Note: For the subsequent library preparation protocols, we recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or H₂O are also acceptable.

1.7. Assess Amplified cDNA Quality and Quantity on a Bioanalyzer

1.7.1. Run 1 µl of amplified cDNA from Step 1.6.20 on a DNA High Sensitivity Chip.

Figure 1.7.1: Example of cDNA size distribution on a Bioanalyzer.