Feb 29, 2024

Public workspacegRNA POOL NGS SEQUENCING LIBRARY PREPARATION

This protocol is a draft, published without a DOI.
  • 1Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 2Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
  • 4School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 5Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 6The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA;
  • 7Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
  • 8The New York Stem Cell Foundation Research Institute, New York, NY, USA
Renuka Ravi Gupta
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Protocol Citation: Renuka Ravi Gupta, Helaine Graziele Santos Vieira, Helen E. King, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Robert J Weatheritt, Lorenz Studer, Joseph Powell 2024. gRNA POOL NGS SEQUENCING LIBRARY PREPARATION. protocols.io https://protocols.io/view/grna-pool-ngs-sequencing-library-preparation-c9wgz7bw
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: In development
We are still developing and optimizing this protocol
Created: February 28, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95912
Keywords: ASAPCRN, CRISPRi machinery, Perturb-Seq, NGS sequencing
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000472
Abstract
Quantification of the frequency of each sgRNA in the library pool is necessary before proceeding to the 10x Genomic assay. We prepare genomic DNA collected after lentiviral transduction of the CRISPR library in H9 dCAS9 cell line to verify the plasmid/viral pool for the CRISPR library via Next Generation Sequencing (NGS sequencing). Genomic DNA was extracted from cells of interest, PCR amplified and the final product was sent to the sequencing facility, where the downstream cleanup and sequencing was performed.
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gRNA POOL NGS SEQUEN...
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Materials
Kits
ABC
KITCOMPANYCATALOG
QIAamp DNA Blood Mini KitQiagen51104
1x dsDNA, high sensitivityThermofisher ScientificQ33231
KAPA HiFi Hotstart PCR KitRocheKK2502
Primers:
The Illumina overhang adapter sequences were added to the locus specific sequences:
  • Forward overhang: 5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACA [locus specific sequence]
  • Reverse overhang: 5’GTCTCGTGGGCTCGGAGATTGTATAAGAGACAG‐[locus specific sequence]
The protospacer site within pCRISPRi_dual guide gRNA library plasmid was amplified by PCR using primers containing Illumina adapters added to the locus specific sequence.
Fig:1 PCR to amplify protospacer A/C = mU6 Fwd +RW704_illuSeq_Rev, the amplicon size= 171 bp
Fig:2 PCR to amplify protospacer B/D = LKO/U6 Fwd + RW705_illuSeq_Rev, the amplicon size 164 bp
PRIMERSEQUENCE
mU6 -Fwd with Fwd overhangTCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CA CAG CAC AAA AGG AAA CTC ACC
RW704 illuSeq-Rev with Rev overhangGTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GCG GCC AAG TTG TAA ACG G
LKO/U6 with Fwd overhangTCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GAC TAT CAT ATG CTT ACC GT
RW705 illuSeq-Rev withReverse overhangGTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G GGC CAA GTT GAT AAC GGA
Table:1 PCR to amplify protospacer B/D = LKO/U6 Fwd + RW705_illuSeq_Rev, the amplicon size 164bp



Before start
H9 dCAS9 CRISPRi were transduced with the pooled CRISPRi library to obtain the gDNA (genomic DNA) after lentiviral transduction.
CITATION
Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell. LENTIVIRAL TRANSDUCTION OF HUMAN PLURIPOTENT STEM CELLS. protocols.io.
LINK
https://protocols.io/view/lentiviral-transduction-of-human-pluripotent-stem-c9wtz7en

DNA Extraction
DNA Extraction
Collect cells after FACS sort in E8 flex media.
Centrifuge the cells at 300 g for 4 mins.
Aspirate the supernatant and freeze down the cell pellet or continue with DNA extraction using the QIAamp DNA Blood Mini Kit.
Measure the concentration of the DNA using nanodrop to check the quality of the DNA and using Qubit to get the precise concentration of the DNA
PCR amplification
PCR amplification
PCR reactions are set up as follows:
  • Template 50 ng pCRISPRi_dual guide gRNA library, 1:1 primers mU6 Fwd + RW704 illuSeq-Rev
  • Template 50 ng pCRISPRi_dual guide gRNA library, 1:1 primers LKO/U6 Fwd + RW705 illuSeq-Rev
  • Inorder to ensure enough yield of DNA product we load 50 ng of DNA/ reaction for the PCR amplification for each run and set up at least 4 PCR reactions which will be pooled to ensure even distribution of the sgRNAs.

ABC
REAGENT1X in uLFINAL CONCENTRATION
2X KAPA HiFi HotStart Ready Mix12.51X
10uM Fwd Primer0.750.3uM
10uM Rev Primer0.750.3uM
Template DNA (50ng)As requiredAs required
PCR grade Water Up to 25uLN/A
Detailed protocol for PCR sample preparation can be found in this link: https://rochesequencingstore.com/wp-content/uploads/2023/02/KAPA-HiFi-HotStart-ReadyMix-PCR-Kit-Technical-Data-Sheet_v14-22.pdf

Set up the thermal cycler using the following program: 
95ºC for 3 minutes
25 cycles of: 
  • 95ºC for 30 seconds
  • 95ºC for 30 seconds
  • 95ºC for 30 seconds
72ºC for 5 mins
Hold at 4ºC
Detailed protocol for PCR sample preparation can be found in this link: https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

Pool the PCR products with similar forward and reverse primers together and send them to the sequencing platform.
QC for the final product was done by the sequencing platform by running the agarose gel to verify the required size of band followed by downstream sequencing.
Protocol references
Citations
Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell. LENTIVIRAL TRANSDUCTION OF HUMAN PLURIPOTENT STEM CELLS
https://protocols.io/view/lentiviral-transduction-of-human-pluripotent-stem-c9wtz7en