Protocol for Cells: Cell Lysis Using NEBNext^® Single Cell Lysis Module (E5530)

	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 input.
	Colored bullets indicate the cap color or label stripe of the reagent to be added to a reaction.

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 module. See Section 1.2 and 1.3 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.

Notes

Keep enzymes on ice.

1.1. Sample and Reagents Preparation

1.1.1. Briefly centrifuge the tube containing Murine RNase Inhibitor to collect solutions to the bottom of the tube, then place on ice.

1.1.2. Thaw the 10X NEBNext Cell Lysis Buffer at room temperature. If it appears cloudy after thawing, incubate briefly at 37°C to

clear up the solution. Leave the 10X NEBNext Cell Lysis Buffer at room temperature.

1.2. Cell Collection (< 1 μl volume) 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 Section 1.3.

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

COMPONENT	VOLUME (μl) PER REACTION
(white) NEBNext Cell Lysis Buffer (10X)	0.5 μl
(white) Murine RNase Buffer	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.

1.2.5. Proceed immediately to cDNA synthesis and amplification using NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB #E6420) or NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (NEB #E6421).

1.3. Cell Collection (≥ 1 μl volume) and Lysis

1.3.1. 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.

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

1.3.2 Add prepared cell lysis buffer to the cells, mix solution thoroughly by pipetting, avoiding bubbles. Centrifuge briefly to collect

solution to the bottom of the tube.

1.3.3. Incubate at room temperature for 5 minutes

1.3.4. Proceed immediately to cDNA synthesis and amplification using NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB #E6420) or NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (NEB #E6421).