Sep 17, 2024

Public workspaceHybrid selection protocol using 10x Single-Cell RNA-Seq assay library

DOI
dx.doi.org/10.17504/protocols.io.x54v924jzl3e/v1
  • 1Broad Institute of MIT and Harvard
xian Adiconis
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DOI: dx.doi.org/10.17504/protocols.io.x54v924jzl3e/v1
Protocol Citation: Xian Adiconis, Joshua Z Levin 2024. Hybrid selection protocol using 10x Single-Cell RNA-Seq assay library. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v924jzl3e/v1
Manuscript citation:
Simmons SK, Adiconis X, Haywood N, Parker J, Lin Z, Liao Z, Tuncali I, Al'Khafaji AM, Shin A, Jagadeesh K, Gosik K, Gatzen M, Smith JT, El Kodsi DN, Kuras Y, Baecher-Allan C, Serrano GE, Beach TG, Garimella K, Rozenblatt-Rosen O, Regev A, Dong X, Scherzer CR, Levin JZ. Experimental and Computational Methods for Allelic Imbalance Analysis from Single-Nucleus RNA-seq Data. bioRxiv [Preprint]. 2024 Aug 16:2024.08.13.607784. doi: 10.1101/2024.08.13.607784. PMID: 39185246; PMCID: PMC11343128.
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: September 10, 2024
Last Modified: September 17, 2024
Protocol Integer ID: 107222
Keywords: single-cell RNA-seq, 10x Chromium, hybrid selection, targeted sequencing
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000301
Abstract
This protocol is for target enrichment of cDNA libraries generated with 10x Genomics single-cell RNA-seq assays. The protocol consists of parts of vendor provided protocols with minor modification. The hybridization and PCR part is based on "CG000059_DemonstratedProtocolExome_RevC" (10x Genomics) and the capture part is based on "SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol Version C3, September 2019" (Agilent).
Materials
Oligos
ABC
Oligo NameVendorSequence
P5 primer IDT5’-AATGATACGGCGACCACCGA-3’
P7 primer IDT5’-CAAGCAGAAGACGGCATACGA-3’
Reagents
ABC
ReagentsVendorPart Number
xGen® Universal Blocking Oligo – TS-p5, 25 rxnIDT1016184
xGen® Universal Blocking Oligo – TS-p7(8nt), 25 rxnIDT1016188
SureSelectXT Reagent kit for 16 samples,includes library prep and enrichment reagents for post-capture processing on the HiSeq platform. Includes indexes 1 - 16.AgilentG9611A
SureSelect Custom Tier3 3Mb-5.9Mb Probe (up to 180k oligos) sufficient for post-capture processing of 16 samples. With the following configuration: Design/ ELID # : S3333112Agilent5191-6910
Dynabeads MyOne Streptavidin T1, 2mlThermo Fisher Scientific65601
Amp Mix10x Genomics220129
Agencourt AMPure® XP SPRI beads, 60 mlBeckman Coulter GenomicsA63881
Safety warnings
For hazard information and safety warnings, please refer to the MSDSs (Material Safety Data Sheets).
Sample preparation
Sample preparation
30m
30m
Use Amount750 ng of cDNA library generated with the 10x Chromium single-cell RNA-seq assay.

Add Amount1 µL each of TS-p5 and TS-p7 blocking oligos.


If the total volume exceeds Amount3.4 µL , use a speed-vac and set the temperature at Temperature30 °C to reduce the volume to the desired volume of Amount3.4 µL .

20m
Buffer and reaction mix preparation
Buffer and reaction mix preparation
20m
20m
Prepare the Hybridization Buffer with reagents from the SureSelectXT kit at TemperatureRoom temperature
Prepare the volume for at least 5 reactions to ensure accurate pipetting.
ABC
1x (µl)5x (µl)
Hyb1 (orange)6.6333.15
Hyb2 (red)0.271.35
Hyb3 (yellow)2.6513.25
Hyb4 (black)3.4517.25
Total 1365
10m
Prepare the block mix at Temperature4 °C
AB
1x (µl)
Indexing Block1 (green)2.5
Block 2 (blue)2.5
H2O0.6
Total5.6
10m
Sample and block denaturing
Sample and block denaturing
5m
5m
Add Amount5.6 µL Block mix to the concentrated Amount3.4 µL sample, mix well and transfer to the 0.2ml PCR tube strip (Axygen), and place into a thermocycler following
Thermocycler Conditions: (Lid@Temperature105 °C , 100 µl)
Temperature95 °C , Duration00:05:00
Temperature65 °C , Duration00:00:00 hold

5m
Hybridization
Hybridization
1d
1d
While step 6 mix is incubated at Temperature65 °C for Duration00:05:00 , prepare the following at TemperatureRoom temperature
AB
1x (µl)
Hybridization Buffer (Step 4)13
25% RNase Block solution (for >= 3Mb)0.5 µl RNase Block (purple)+1.5 µl H2O=2 ul
Capture library(>=3 Mb)5
Total 20
Mix well by high speed vortexing for Duration00:00:05 , spin down briefly, add into the sample and block mix at Temperature65 °C , mix by pipetting 8-10x, use a new cap strip to seal the tubes
Incubate at Temperature65 °C for Duration16:00:00 to Duration24:00:00

1d
Library capture
Library capture
2h
2h
In a strip tube, use Amount50 µL MyOne Streptavidin T1 beads per sample, wash with Amount200 µL SureSelect Binding buffer 3 times, resuspend in Amount200 µL SureSelect Binding buffer.


15m
Bring the washed beads from step 8 near the thermocycler, add the Temperature65 °C reaction mix (~Amount27 µL left) right into the Amount200 µL beads, gentle pipetting mix, incubate at TemperatureRoom temperature for Duration00:30:00 , pipetting mix 6 times every Duration00:05:00

35m
Place on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear. Resuspend the beads in Amount200 µL SureSelect Wash buffer 1, incubate at TemperatureRoom temperature for Duration00:15:00

15m
Meanwhile, pre-warm SureSelect Wash buffer 2 at Temperature65 °C in strip tubes with Amount200 µL per well and 3 wells per reaction on a 96-well heating block (or a thermocycler with reaction volume capacity of Amount200 µL )

5m
Place step 10 reaction mix on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear.
Resuspend the beads in Amount200 µL pre-warmed SureSelect Wash buffer 2, incubate at Temperature65 °C for Duration00:10:00 Place the reaction mix on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear.
15m
Repeat step 13 two more times and total three washes with pre-warmed SureSelect Wash buffer 2. And make sure all the wash buffer has been removed in the final wash.

30m
Resuspend the beads in Amount30 µL H2O and keep on ice.

5m
Enrichment PCR
Enrichment PCR
2h
2h
On ice, assemble the following mix
AB
Amp Mix50
P5 primer (10µM)2
P7 primer (10µM)2
*cDNA containing beads30
H2O16
Total100
Thermocycler Conditions: (Lid@Temperature105 °C , 100 µl)
Temperature98 °C ,Duration00:00:45
then 9 cycles of
Temperature98 °C , Duration00:00:15
Temperature60 °C , Duration00:00:30
Temperature72 °C , Duration00:00:30
then
Temperature72 °C , Duration00:01:00
Temperature4 °C , Duration00:00:00 hold

30m
Place the PCR reaction tube on a magnet stand, wait until it clears and recover Amount100 µL supernatant to a new PCR tube strip.

5m
Add Amount180 µL SPRI beads (1.8x) to the recovered PCR product, mix well by pipetting, pellet the beads on a magnet stand, after supernatant removal, wash with Amount300 µL 80% ethanol twice, elute with Amount20 µL buffer EB (10 mM Tris-HCl, pH 8.5).

15m
QC with Quant-it (Thermo Fisher Scientific) and BioAnalyzer DNA HS assay (Agilent).
1h
Protocol references
1. "CG000059_DemonstratedProtocolExome_RevC" https://assets.ctfassets.net/an68im79xiti/Zm2u8VlFa8qGYW4SGKG6e/4bddcc3cd60201388f7b82d241547086/CG000059_DemonstratedProtocolExome_RevC.pdf

2. "SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol Version C3, September 2019"
newer version at https://www.agilent.com/cs/library/usermanuals/public/G7530-90000.pdf