FACS was performed on the FACSAria III platform (BD) at DFCI Flow-core facility. For GFP labelled cell collection, 488 nm laser and 585/42 filter were used. For mKate2 651 nm laser and 610/20 filter were used. For single channels, 4 fractions were collected based on histogram profile, using 4-way sorting, approximately 25% per each of under curve area. We named these fractions: low (0–25%), mid low (25–50%), mid high (50%–75%), high (75–100%). For dual reporter, 4 fractions were collected, 2.5% of each, including dual negative, dual positive, GFP positive and mKate2 positive fractions. Cells were collected into 5 ml tubes. After collecting cell pellet, direct PCR was performed using Phire Tissue master mix (F170L, Thermo), according to the manufacturer recommendations. Guide RNA abundance was determined from each fraction by deep amplicon sequencing. Library preparation was performed according to the Broad Institute recommended protocol at the GPP portal using Argon primers for the demultiplexing. (https://portals.broadinstitute.org/gpp/public/resources/protocols )
Phire tissue master mix
Manufactured by Thermo Fisher Scientific
Phire Tissue master mix is a ready-to-use solution for PCR amplification of DNA from various tissue samples. It contains all the necessary components, including a thermostable DNA polymerase, for efficient and reliable DNA amplification.
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Lab products found in correlation
2 protocols using phire tissue master mix
1
FACS-based Cell Fractionation and Sequencing
2
Quantitative CRISPR Guide RNA Profiling
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FACS was performed on the FACSAria III platform (BD) at DFCI Flow-core facility. For GFP labeled cell collection, 488 nm laser and 585/42 filter were used. For mKate2 651 nm laser and 610/20 filter were used. For single channels, 4 fractions were collected based on histogram profile, using 4-way sorting, approximately 25% per each of under curve area. We named these fractions: low (0–25%), mid low (25–50%), mid high (50–75%), high (75–100%). For dual reporter, 4 fractions were collected, 2.5% of each, including dual negative, dual positive, GFP positive and mKate2 positive fractions. Cells were collected into 5-ml tubes. After collecting cell pellet, direct PCR was performed using Phire Tissue master mix (F170L, Thermo), according to the manufacturer recommendations. Guide RNA abundance was determined from each fraction by deep amplicon sequencing. Library preparation was performed according to the Broad Institute recommended protocol at the GPP portal using Argon primers for the demultiplexing. (https://portals.broadinstitute.org/gpp/public/resources/protocols ).
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