Dec 01, 2021
Part 1: SmartSeq
- cecilia1,
- Suzie Alarcon1,
- Alessandro Sette1
- 1La Jolla Institute for Immunology
Protocol Citation: cecilia, Suzie Alarcon, Alessandro Sette 2021. Part 1: SmartSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bxr3pm8n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: August 27, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 52763
Keywords: SmartSeq, Anneal primers, ASAPCRN
Abstract
This protocol details the procedure of SmartSeq.
Attachments
d2bybg4xf.docx
27KB
Materials
Materials:
oligo-dT primer (10 micromolar (µM) )
dNTP mix (10 millimolar (mM) )
SuperScriptII reverse transcriptase (200 U/µL )
RNAse inhibitor (40 U/µL )
SuperscriptII first-strand buffer (5x)
DTT (100 millimolar (mM) )
Betaine(5 Molarity (M) )
Nuclease-free H2O
TSO(100 micromolar (µM) )
First-strand rxn (previous step)
Kapa HiFi HotStart ReadyMix (2x)
IS PCR primers (10 micromolar (µM) )
H2O
INPUT:
INPUT:
Use total RNA, ranging between 10 pg -30 pg up to 10 ng . 2.6 µL will be used per sample.
ANNEAL PRIMERS:
ANNEAL PRIMERS:
8m
8m
On ice , add each sample (2.6 µL total RNA) to a thin-walled 0.2 mL PCR tube and add:
| A | B | C | D | |
| Item | Volume (uL) | ______xMM | Lot# | |
| oligo-dT primer (10uM) | 1 | |||
| dNTP mix (10mM) | 1 |
Centrifuge at 700 x g for 00:00:10 at Room temperature . Incubate @72 °C for 00:03:00 , spin down, put On ice .
3m 10s
REVERSE TRANSCRIPTION: add the following On 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 15 µL 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 25 µL beads to each sample).
Allow beads to incubate with sample for 00:05:00 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.
5m
Carefully pipette 150 µL -200 µL of freshly prepared 80% EtOH to the supernatant and incubate for 00:00:30 . Remove EtOH and immediately repeat this step.
30s
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.
Note
Do not overdry, but do not allow excess EtOH to remain on beads. This usually takes about 2 minutes, depending on ambient environment.
Elute with 17.5 µL H2O, keep 15 µL . Once beads are dry, resuspend in eluate and incubate for 00:02:00 - 00:05:00 . Place slurry on magnet and allow supernatant to clear. Once all beads are on the magnet, carefully remove 15 µL of the clear eluate.
7m
Quant the cleaned cDNA on Tapestation, using D5000 reagents or HSD5000, depending on expected yield and according to manufacturers suggestions.