Oct 01, 2024
Version 1
MAS-ISO-seq - from 10x Single Cell Gene Expression Libraries V.1
- Aziz Al'Khafaji1
- 1Broad Institute
Protocol Citation: Aziz Al'Khafaji 2024. MAS-ISO-seq - from 10x Single Cell Gene Expression Libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p5ezl25/v1
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
We use this protocol and it's working
Created: December 31, 2021
Last Modified: October 01, 2024
Protocol Integer ID: 56468
Abstract
Method for MAS-ISO-seq from 10x Single Cell Gene Expression Libraries.
Materials
Reagents:
2x Kapa HiFi Uracil+ ReadyMix (Roche #7959079001)
SPRIselect (Beckman Coulter B23318)
Qubit (Thermo #Q32851)
Dynabeads™ kilobaseBINDER™ (Thermo #60101)
Low-EDTA TE (1X), pH 8.0 (VWR 10128-588)
USER® Enzyme (M5505S)
HiFi Taq DNA Ligase (M0647S)
Genomic DNA ScreenTape/Reagent
Primers:
| A | B | C | |
| cDNA_amp_primers | |||
| 5' Libraries | |||
| Fwd_R1_10x5'_PT_MAS | CTACACGACGCTCTTCCGAT*C*T | AAO272 | |
| Rev_10x5_dUbio_PT | /5Biosg/UAUAAGCAGTGGTATCAACGCAG*A*G | AAO273 | |
| 3' Libraries | |||
| dUbio_venus_PT | /5Biosg/UAUCTACACGACGCTCTTCCGAT*C*T | AAO365 | |
| mars_PT | AAGCAGTGGTATCAACGCAG*A*G | AAO366 |
| A | B | C | |
| MAS-seq primer plate | |||
| Well Position | Name | Sequence | |
| A1 | *A-Fwd_5'_10x* | AGCTTACTTGTGAAGATCTACACGACGCTCTTCCGATCT | |
| A2 | B-Fwd_5'_10x | ACTTGTAAGCUGTCTAUCTACACGACGCTCTTCCGATCT | |
| A3 | C-Fwd_5'_10x | ACTCTGUCAGGTCCGAUCTACACGACGCTCTTCCGATCT | |
| A4 | D-Fwd_5'_10x | ACCTCCTCCUCCAGAAUCTACACGACGCTCTTCCGATCT | |
| A5 | *E*-Fwd_5'_10x | AACCGGACACACUTAGUCTACACGACGCTCTTCCGATCT | |
| A6 | F-Fwd_5'_10x | AGAGTCCAAUTCGCAGUCTACACGACGCTCTTCCGATCT | |
| A7 | G-Fwd_5'_10x | AATCAAGGCUTAACGGUCTACACGACGCTCTTCCGATCT | |
| A8 | H-Fwd_5'_10x | ATGTTGAAUCCTAGCGUCTACACGACGCTCTTCCGATCT | |
| A9 | I-Fwd_5'_10x | AGTGCGTUGCGAATTGUCTACACGACGCTCTTCCGATCT | |
| A10 | J-Fwd_5'_10x | AATTGCGUAGTTGGCCUCTACACGACGCTCTTCCGATCT | |
| A11 | K-Fwd_5'_10x | ACACTTGGUCGCAATCUCTACACGACGCTCTTCCGATCT | |
| A12 | L-Fwd_5'_10x | AGTAAGCCUTCGTGTCUCTACACGACGCTCTTCCGATCT | |
| B1 | M-Fwd_5'_10x | ACCTAGAUCAGAGCCTUCTACACGACGCTCTTCCGATCT | |
| B2 | N-Fwd_5'_10x | AGGTAUGCCGGUTAAGUCTACACGACGCTCTTCCGATCT | |
| B3 | O-Fwd_5'_10x | AAGUCACCGGCACCUTUCTACACGACGCTCTTCCGATCT | |
| C1 | B-Rev_5'_10x | ATAGACAGCUTACAAGUAAGCAGTGGTATCAACGCAGAG | |
| C2 | C-Rev_5'_10x | ATCGGACCUGACAGAGUAAGCAGTGGTATCAACGCAGAG | |
| C3 | D-Rev_5'_10x | ATTCUGGAGGAGGAGGUAAGCAGTGGTATCAACGCAGAG | |
| C4 | *E*-Rev_5'_10x | ACTAAGTGUGTCCGGTUAAGCAGTGGTATCAACGCAGAG | |
| C5 | F-Rev_5'_10x | ACTGCGAAUTGGACTCUAAGCAGTGGTATCAACGCAGAG | |
| C6 | G-Rev_5'_10x | ACCGTUAAGCCTTGATUAAGCAGTGGTATCAACGCAGAG | |
| C7 | H-Rev_5'_10x | ACGCTAGGAUTCAACAUAAGCAGTGGTATCAACGCAGAG | |
| C8 | I-Rev_5'_10x | ACAATUCGCAACGCACUAAGCAGTGGTATCAACGCAGAG | |
| C9 | J-Rev_5'_10x | AGGCCAACUACGCAATUAAGCAGTGGTATCAACGCAGAG | |
| C10 | K-Rev_5'_10x | AGATUGCGACCAAGTGUAAGCAGTGGTATCAACGCAGAG | |
| C11 | L-Rev_5'_10x | AGACACGAAGGCUTACUAAGCAGTGGTATCAACGCAGAG | |
| C12 | M-Rev_5'_10x | AAGGCTCUGATCTAGGUAAGCAGTGGTATCAACGCAGAG | |
| D1 | N-Rev_5'_10x | ACTUAACCGGCAUACCUAAGCAGTGGTATCAACGCAGAG | |
| D2 | O-Rev_5'_10x | AAAGGUGCCGGUGACTUAAGCAGTGGTATCAACGCAGAG | |
| D3 | *P-Rev_5'_10x* | ATCTCGAGCCACTTCATAAGCAGTGGTATCAACGCAGAG |
Mix MAS primer pairs:
Order primers reconstituted as 100µM
Mix equal volumes of primer pairs according to the table below to create a 50µM MAS primer mix plate.
Create a 5µM working MAS-primer plate by diluting 10-fold in low-TE.
| A | B | C | |
| MAS-seq primer mixing strategy | |||
| wells | adapter pairs | ||
| 1 | A1 & C1 | *A|B | |
| 2 | A2 & C2 | B|C | |
| 3 | A3 & C3 | C|D | |
| 4 | A4 & C4 | D|E | |
| 5 | A5 & C5 | E|F | |
| 6 | A6 & C6 | F|G | |
| 7 | A7 & C7 | G|H | |
| 8 | A8 & C8 | H|I | |
| 9 | A9 & C9 | I|J | |
| 10 | A10 & C10 | J|K | |
| 11 | A11 & C11 | K|L | |
| 12 | A12 & C12 | L|M | |
| 13 | B1 & D1 | M|N | |
| 14 | B2 & D2 | N|O | |
| 15 | B3 & D3 | O|P* | |
WTA amplification:
Set up the following reactions on ice
For 5' libraries:
| A | B | C | |
| Reagent | Reaction conc. | µL per. reaction | |
| Nuclease Free Water | 35 | ||
| Kapa HiFi Uracil+ ReadyMix (2X) | 1X | 50 | |
| Fwd_R1_10x5'_PT_MAS AAO272(10uM) | 0.5 µM | 5 | |
| Rev_10x5_dUbio_PT AAO273(10uM) | 0.5 µM | 5 | |
| 10x 5' cDNA library (whole transcriptome amplification); 2-5ng/µL | 5 | ||
| Total | 100 µL |
For 3' libraries:
| A | B | C | D | |
| Reagent | Reaction conc. | µL per. reaction | ||
| Nuclease Free Water | 35 | |||
| Kapa HiFi Uracil+ ReadyMix (2X) | 1X | 50 | ||
| Fwd_R1_10x5'_PT_MAS AAO365(10uM) | 0.5 µM | 5 | ||
| Rev_10x5_dUbio_PT AAO366(10uM) | 0.5 µM | 5 | ||
| 10x 3' cDNA library (whole transcriptome amplification); 2-5ng/µL | 5 | |||
| Total | 100 µL |
| A | B | C | D | E | |
| Step | Temperature | Time | Cycles | ||
| Initial denaturation | 98 °C | 3 min | 1x | ||
| Denaturation | 98 °C | 20 sec | 5x | ||
| Annealing | 65°C | 30 sec | |||
| Elongation | 72 °C | 8 min | |||
| Final Elongation | 72 °C | 10 min | 1x | ||
| Hold | 4 °C | Hold |
Reaction cleanup and quantification
- 0.8x SPRIselect cleanup - 80 µL beads in 100 µL PCR reaction from step 2.
- Elute in 46 µL low-TE
- Qubit quantification
TSO artifact removal
- Transfer 10 μL (100 μg) resuspended Dynabeads™ kilobaseBINDER™ streptavidin beads to a PCR tube.
- Place the tube on the magnet for 2 min.
- Carefully remove and discard the supernatant while the tube remains on the magnet. Avoid touching the bead pellet with the pipette tip.
- Remove the tube from the magnet. Add 40 μL Binding Solution along the inside wall of the tube where the beads are collected and gently resuspend by pipetting. Note: the solution may be viscous. Avoid foaming.
- Place the tube on the magnet for 2 min and remove the supernatant.
- Resuspend the beads in 40 μL Binding Solution.
- Add 40 μL of a solution containing the biotinylated DNA-fragments to the resuspended beads. Mix carefully to avoid foaming of the solution.
- Incubate the tube at room temperature for 3 hours on a roller to keep the beads in suspension.
- Place the tube on the magnet and remove the supernatant as in step 3, above.
- Wash the Dynabeads®/DNA-complex 2x in 80 μL Washing Solution and once in distilled water.
- Resuspend the Dynabeads®/DNA-complex in 40ul Low-TE.
- Add 2ul USER and incubate in a rotator at 37C for 2 hours.
- Place the tube on the magnet and move the supernatant containing the library to a fresh tube.
- Cleanup - 0.8x SPRI (32 µL beads in 40 µL library)
- Elute in 46 µL low-TE.
- Qubit quantification
MAS adapter PCR
Set up all reactions on ice
- Create the following master mix:
| A | B | C | D | E | F | |
| Reagent | Reaction conc. | µL per. reaction | ||||
| Nuclease Free Water | 618.7 | |||||
| Kapa HiFi Uracil+ ReadyMix (2X) | 1X | 800 | ||||
| Purified cDNA from step 4 | 21.3 | |||||
| Total | 1440 µL |
2. Distribute 90 µL of Master Mix into each of 15 PCR tubes
3. Distribute 10 µL 5 µM MAS-seq primer pair mix into each of 15 PCR tubes
Cycling conditions:
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Initial denaturation | 98 °C | 3 min | 1x | |
| Denaturation | 98 °C | 20 sec | *n*x | |
| Annealing | 65°C | 30 sec | ||
| Elongation | 72 °C | 8 min | ||
| Final Elongation | 72 °C | 10 min | 1x | |
| Hold | 4 °C | Hold |
Note - optimal cycle number is a function of amount of input material after TSO artifact removal. See table below for cycle determination
| A | B | |
| cDNA concentration (ng/µL) | cycle number | |
| 1 - 2.5 | 10 | |
| 2.5 - 5 | 9 | |
| 5 - 7.5 | 8 |
Reaction cleanup
- Pool 15 x 95 µL reactions in 5ml tube. (Note* attaining equimolar amounts of each adapter PCR is key to efficient array assembly - be mindful to add the same amount of each adapter PCR to the pool. To account for handling, 95/100 µL of the PCR is advised to be pooled from each reaction.)
- SPRIselect cleanup - 0.7x (997.5 µL beads in 1425 µL pooled MAS adapter PCR)
- Elute in 450 µL low TE buffer.
- Qubit quantify
- Move 435 µL of the library to a 1.5 mL microtube tube and added 15 µL of USER enzyme. (Note* 10-15 µg is advised going into this step. Dilute going into this step if sample is too concentrated.)
- Incubate reaction at 37 for 2.5 hours
- Add 51 µL Hifi Ligase buffer and 15ul Hifi ligase to the USER reaction.
- Distribute to 5x PCR tubes and set in thermocycler at 42C for 2 hours.
- Pool reaction into a 1.5 mL microtube using a wide bore tip
- SPRIselect cleanup - 0.7x ( 361.2 µL beads in 516 µL reaction), mix gently with wide bore tip. Set on rotator for 5 min.
- Incubate at 37C for 10min - elute in 180 µL low TE.
- Qubit quantify
MAS library quantification
Quantify MAS arrays with Genomic DNA ScreenTape or Femto Pulse.
See example below:
Optional Size Selection
SMRTbell Express Template Prep: Use this as input material for PacBio SMRTbell Express Template Prep Kit 2.0, starting at the " Remove ssDNA Overhangs" step (page 8: https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-HiFi-SMRTbell-Libraries-using-SMRTbell-Express-Template-Prep-Kit-2.0.pdf)
Minimum starting material going into the PacBio SMRTbell Express Template Prep Kit 2.0 : 5µg