Nov 07, 2019
Massively parallel long-read sequencing of single cell RNA isoforms
- Jafar S Jabbari1,
- Luyi Tian2
- 1The Walter and Eliza Hall Institute of Medical Research;
- 2Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- Human Cell Atlas Method Development Community
Protocol Citation: Jafar S Jabbari, Luyi Tian 2019. Massively parallel long-read sequencing of single cell RNA isoforms. protocols.io https://dx.doi.org/10.17504/protocols.io.8d9hs96
Manuscript citation:
This protocol currently has not been linked to any publication.
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: October 18, 2019
Last Modified: November 07, 2019
Protocol Integer ID: 28833
Keywords: 10x Genomics GEM Subsampling, Single Cell Full-length cDNA Sequencing, Single Cell Isoform Sequencing, Nanopore, PacBio,
Abstract
This protocol describes the subsampling of 10x Chromium generated single cell GEMs after reverse transcription for cDNA amplification. The protocol enables transcriptome sequencing of full-length cDNA from a flexible number of single cells captured on Chromium using long-read sequencers such as Nanopore and PacBio, in addition to preparing short-read Illumina libraries. The protocol is compatible with 10x Single Cell 3' and 5' Gene Expression kits including feature barcoding and also other 10x compatible multiomics methodologies such as CITE-seq.
Guidelines
This protocol assumes the operator has access to a 10x Genomics Chromium Controller and expertise in generating single cell 3' or 5' libraries using 10x single cell transcriptomics kits.
Materials
| Name | Catalog# | Vendor | |
| Chromium Chip B Single Cell Kit | 1000074 | 10x Genomics | |
| Or Chromium Single Cell A Chip Kit | 1000009 | 10x Genomics | |
| Chromium Single Cell 3′ Library & Gel Bead Kit v3 | 1000092 | 10x Genomics | |
| Chromium Single Cell 3′ Library Construction Kit v3 | 1000078 | 10x Genomics | |
| Or Chromium Single Cell 5′ Library & Gel Bead Kit | 1000014 | 10x Genomics | |
| Chromium Single Cell 5’ Library Construction Kit | 1000020 | 10x Genomics | |
| PCR Tubes 0.2 ml 8-tube strips | 951010022 | Eppendorf | |
| DNA LoBind Tubes, 1.5 ml | 022431021 | Eppendorf | |
| Tips LTS 200UL Filter RT-L200FLR | 30389240 | Rainin | |
| Tips LTS 20UL Filter RT-L10FLR | 30389226 | Rainin | |
| Nuclease-free Water | AM9937 | Thermo Fisher Scientific | |
| Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) | 12090-015 | Thermo Fisher Scientific | |
| Ethanol, Pure (200 Proof, anhydrous | E7023-500ML | Millipore Sigma | |
| SPRIselect Reagent Kit | B23318 | Beckman Coulter | |
| 10% Tween 20 | 1662404 | Bio-Rad | |
| Glycerin (glycerol), 50% (v/v) Aqueous Solution | 3290-32 | Ricca Chemical Company | |
| Qiagen Buffer EB | 19086 | Qiagen | |
| High Sensitivity D1000 ScreenTape/Reagents | 5067-5592/ 5067-5593 | Agilent | |
| High Sensitivity D5000 ScreenTape/Reagents | 5067-5584/ 5067-5585 | Agilent | |
| Qubit dsDNA HS Assay Kit | Q32854 | Thermo Fisher Scientific | |
| PrimeSTAR® GXL DNA Polymerase | R050B | Takara |
| Primer | Sequence | Supplier | |
| FPSfiIA | 5’-ACTAAAGGCCATTACGGCCTACACGACGCTCTTCCGATCT-3' | Oligo vendor | |
| RPSfiIBr | 5’-TTACAGGCCGTAATGGCCAAGCAGTGGTATCAACGCAGAGTA-3' | Oligo vendor |
Before start
It is recommended that the operator reviews the corresponding User Guide for the 10x Genomics kit being used.
CG000183_ChromiumSingleCell3__v3_UG_RevB.pdf CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_C.pdf CG000086_ChromiumSingleCellV_D_J_ReagentKits_UG_RevL.pdf CG000207_ChromiumNextGEMSingleCellV_D_J_ReagentKits_v1.1_UG_RevD.pdf