Part 1: SmartSeq

Use total RNA, ranging between 10pg-30pg up to 10ng. 2.6µL will be used per sample.

On ice, add each sample (2.6µL total RNA) to a thin-walled 0.2mL PCR tube and add:

A	B	C	D
Item	Volume (uL)	______xMM	Lot#
oligo-dT primer (10uM)	1
dNTP mix (10mM)	1

Centrifuge at 700x g,0h 0m 0s for 0h 0m 10s at Room temperature. I ncubate @ for 72°C for 0h 3m 0s, spin down, put 72On ice.

REVERSE TRANSCRIPTION: add the following 72On ice:

A	B	C	D
Item	Volume(uL)	______xMM	Lot#
SuperScriptII reverse transcriptase (200U/uL)	0.50
RNAse inhibitor (40U/uL)	0.25
SuperscriptII first-strand buffer (5x)	2
DTT (100mM)	0.50
Betaine(5M)*	2
Nuclease-free H2O	0.06
TSO(100uM)**	0.10
Total volume:	5.41		N/A

*stored at 4oC.**template-switching oligos, stored in -80oC.

Mix, spin down, and incubate in thermocycler on following settings:

A	B	C
Step	Temp(C)	Time(hh:mm:ss)
1	42	01:30:00
2	50	00:02:00
3	42	00:02:00
4	Return to step 2, 5x
5	70	00:15:00
6	4	hold

PCR PRE-AMPLIFICATION: Prepare Master Mix during Reverse Transcription rxn.

A	B	C	D
Item	Volume(uL)	______xMM	Lot#
First-strand rxn (previous step)	10		N/A
Kapa HiFi HotStart ReadyMix (2x)	12.5
IS PCR primers (10uM)	0.25
H2O	2.25
Total volume:	25		N/A

Add 15µL PreAmp Master Mix to each sample, seal, mix, spin down, and incubate in thermocycler on following settings:

A	B	C
Step	Temp(C)	Time(hh:mm:ss)
1	98	00:03:00
2	98	00:00:20
3	67	00:00:15
4	72	00:06:00
5	Return to step 2	17x (18 cycles total)
6	72	00:05:00
7	4	hold

Do a standard Ampure or equivalent 1X (1:1 ratio bead to sample) Bead cleanup (add 25µL beads to each sample).

Allow beads to incubate with sample for 0h 5m 0s at Room temperature. After 5 minutes, place samples on magnet until the supernatant is clear and all beads are on the wall of the tube. Carefully remove the supernatant and immediately move to wash steps.

Carefully pipette 150µL-200µL of freshly prepared 80% EtOH to the supernatant and incubate for 0h 0m 30s. Remove EtOH and immediately repeat this step.

80% EtOH wash 2. Once second EtOH wash is completed and EtOH has been removed from the beads, allow the beads to dry until they look glossy.

Elute with 17.5µL H2O, keep 15µL. Once beads are dry, resuspend in eluate and incubate for 0h 2m 0s - 0h 5m 0s. Place slurry on magnet and allow supernatant to clear. Once all beads are on the magnet, carefully remove 15µL of the clear eluate.

Quant the cleaned cDNA on Tapestation, using D5000 reagents or HSD5000, depending on expected yield and according to manufacturers suggestions.