Fragmentation, End Repair & A-tailing

Prepare a thermal cycler with the following incubation protocol:

A	B	C
Lid Temperature	Reaction Volume	Run Time
65°C	50 µl	~35 min
Step	Temperature	Time
Pre-cool block	4°C	Hold
Fragmentation	32°C	5 min
End Repair & A-tailing	65°C	30 min
Hold	72°C	Hold

Thermocycler protocol.

Vortex fragmentation buffer. Verify there is no precipitate.

Prepare fragmentation mix On ice. Pipette mix and centrifuge briefly.

Fragmentation Mix

A	B	C	D
Reagents	1X (μl)	4X+10% (μl)	8X+10% (μl)
Fragmentation Buffer	5	22	44
Fragmentation Enzyme	10	44	88
Total	15	66	132

Calculation for the Fragmentation Mix preparation.

Transfer ONLY 10µLpurified cDNA sample from pellet cleanup to a tube strip. The remaining 30µL(75%) cDNA sample can be stored at 4°C for up to 72h 0m 0s or at -20°C for up to 4 weeks for generating additional 3ʹ Gene Expression libraries.

Add 25µL buffer EB to each sample.

Add 15µL fragmentation mix to each sample.

Pipette mix 15× (pipette set to 35 µl)On ice. Centrifuge briefly.

Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.

Post Fragmentation,End Repair & A-tailing Double Sided Size Selection – SPRIselect:

Vortex to resuspend SPRIselect reagent. Add 30µL SPRIselect (0.6X) reagent to each sample. Pipette mix 15× (pipette set to 75 µl).

Remove the ethanol.

Repeat sub-steps 2.9 and 2.10 for a total of 2 washes.

Centrifuge briefly. Place on the magnet. Low until the solution clears. Remove remaining ethanol.

Remove from the magnet. Add 50.5µL buffer EB to each sample. Pipette mix 15× (pipette set to 45 µl).

Incubate 0h 2m 0s at Room temperature.

Place on the magnet. High until the solution clears.

Transfer50µL sample to a new tube strip.

Incubate 0h 5m 0s at Room temperature.

Place on the magnet. High until the solution clears. DO NOT discard supernatant.

Transfer75µLsupernatant to a new tube strip.

Vortex to resuspend SPRIselect reagent. Add 10µLSPRIselect reagent (0.8X) to each transferred supernatant. Pipette mix 15× (pipette set to 80 µl).

Incubate 0h 5m 0s at Room temperature.

Place on the magnet. High until the solution clears.

Remove 80µL supernatant. DO NOT discard any beads.

Add 125µL 80% ethanol to the pellet. Wait 0h 0m 30s.

Adaptor Ligation

Prepare adaptor ligation Mix. Pipette mix and centrifuge briefly.

Adaptor Ligation Mix:

A	B	C	D
Reagents	1X (μl)	4X+10% (μl)	8X+10% (μl)
Ligation Buffer	20	88	176
DNA Ligase	10	44	88
Adaptor Oligos	20	88	179
Total	50	220	440

Add 50µL adaptor ligation mix to 50µL sample. Pipette mix 15× (pipette set to 90 µl). Centrifuge briefly.

Incubate in a thermal cycler with the following protocol:

A	B	C
Lid Temperature	Reaction Volume	Run Time
30°C	100 µl	15 min
Step	Temperature	Time
1	20°C	15 min
2	4°C	Hold

Post Ligation Cleanup – SPRIselect:

Vortex to resuspend SPRIselect reagent. Add 80µLSPRIselect (0.8X) reagent to each sample. Pipette mix 15× (pipette set to 75 µl).

Remove from the magnet. Add 30.5µL buffer EB to each sample. Pipette mix 15x.

Incubate 0h 2m 0satRoom temperature.

Place on the magnet. Low until the solution clears.

Transfer 30µL sample to a new tube strip.

Incubate 0h 5m 0s at Room temperature.

Place on the magnet. High until the solution clears.

Remove the supernatant.

Add 200µL 80% ethanol to the pellet. Wait 0h 0m 30s.

Remove the ethanol.

Repeat sub-steps 4.5 and 4.6 for a total of 2 washes.

Centrifuge briefly. Place on the magnet. Low until the solution clears.

Remove remaining ethanol. Air dry for 0h 2m 0s.

Sample Index PCR

Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10× Sample Index name (PN-3000431 Dual Index Plate TT Set A well ID) used.

Add 50µL Amp mix to 30µL sample.

Add 20µL of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5× (pipette set to 90 µl). Centrifuge briefly.

Incubate in a thermal cycler with the following protocol:

A	B	C
Lid Temperature	Reaction Volume	Run Time
105°C	100 µl	~25-40 min
Step	Temperature	Time
1	98°C	45 sec
2	98°C	20 sec
3	54°C	30 sec
4	72°C	20 sec
5	Go to Step 2 – * # cycles calculated below
6	72°C	1 min
7	4°C	Hold

A	B
cDNA Input	Total cycles
0.25-25 ng	14-16
25-150 ng	Dec-14
150-500 ng	10-Dec
500-1,000 ng	08-Oct
1,000-1,500 ng	06-Aug
>1,500 ng	5

Store at 4°C for up to 72h 0m 0s or proceed to the next step.

Post Sample Index PCR Double Sided Size Selection – SPRIselect

Vortex to resuspend SPRIselect reagent. Add 60µL SPRIselect (0.6X) reagent to each sample. Pipette mix 15× (pipette set to 150 µl).

Remove the ethanol.

Repeat sub-steps 6.9 and 6.10 for a total of 2 washes.

Centrifuge briefly. Place on the magnet. Low until the solution clears. Remove remaining ethanol.

Remove from the magnet. Add 35.5µL buffer EB to each sample. Pipette mix 15× (pipette set to 35 µl).

Incubate 0h 2m 0s at Room temperature.

Place on the magnet. Low until the solution clears.

Transfer 35µL sample to a new tube strip.

Store at 4°C for up to 72h 0m 0s or at -20°C for long-term storage.

Incubate 0h 5m 0s at Room temperature.

Place on the magnet. High until the solution clears. DO NOT discard supernatant.

Transfer 150µL supernatant to a new tube strip.

Vortex to resuspend SPRIselect reagent. Add 20µL SPRIselect reagent (0.8X)to each transferred supernatant. Pipette mix 15× (pipette set to 150 µl).

Incubate 0h 5m 0s at Room temperature.

Place on the magnet. High until the solution clears.

Remove 165µL supernatant. DO NOT discard any beads.

Add 200µL 80% ethanol to the pellet. Wait 0h 0m 30s.

Post Library Construction QC

Run 1µL sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.

Determine the average fragment size from the Bioanalyzer trace. This will be used as the insert size for library quantification.

3’ Gene Expression Library Sequencing Depth & Run Parameters:

A	B
Sequencing Depth	Minimum 20,000 read pairs per cell
Sequencing Type	Pair-end, dual indexing
Sequencing Read	Recommended Number of cycles
Read 1	28 cycles
i7 Index	10 cycles
i5 Index	10 cycles
Read 2	90 cycles

Once quantified and normalized, the 3’ Gene Expression libraries should be denatured and diluted as recommended for Illumina sequencing platforms.