Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized U87MG or eHAP cells after live/dead selection with Reovirus Type 3 Dearing Strain.

ABSTRACT: eHAP or U87MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million eHAP or U87MG cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. Twenty T175 flasks were used for the U87MG-based screen, while twelve T175 flasks were used for the eHAP-based screen. The cells were allowed to recover for 48 hours before infecting with ReoT3D at an MOI of 0.1. Obvious CPE was observed within 72 hours. eHAP-resistant colonies were harvested two weeks later, while U87MG-resistant colonies were harvested six weeks later. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.

INSTRUMENT(S): HiSeq X Ten

ORGANISM(S): Homo sapiens

SUBMITTER: Jan Carette

PROVIDER: E-MTAB-11158 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Richards Christopher M CM Jabs Sabrina S Qiao Wenjie W Varanese Lauren D LD Schweizer Michaela M Mosen Peter R PR Riley Nicholas M NM Klüssendorf Malte M Zengel James R JR Flynn Ryan A RA Rustagi Arjun A Widen John C JC Peters Christine E CE Ooi Yaw Shin YS Xie Xuping X Shi Pei-Yong PY Bartenschlager Ralf R Puschnik Andreas S AS Bogyo Matthew M Bertozzi Carolyn R CR Blish Catherine A CA Winter Dominic D Nagamine Claude M CM Braulke Thomas T Carette Jan E JE

Science (New York, N.Y.) 20221007 6615

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme traff ...[more]

PMID: 36074821

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Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), were validate with Illumina HumanHap550-Duo Genotyping Beadchip. As seen in (Suppl. Table 5a), the call rates (97.30% to 99.07%) and accuracy or concordance ( > 99.85% for the SNPs called in both amplicon and natural reference) for 5 ng derived amplicons with both Wpa and Gv2 were close to each other and close to native gDNA (call rate: 98.3% to 99.75%). These call rates were better than a recent report (amplicon 95.9% vs. un-amplified 98.5%), in which the early kit Repli-g 625S was applied, and re-genotyping was performed when the performance was low and duplicate samples were filtered for the highest call rate. The genotyping accuracy of Wpa was actually in the same range as the variation in technical replicates with similar SNP typing arrays (99.87% and 99.88%, replicated Affymetrix array, or between Affymetrix and Illumina arrays). Importantly, the genotyping concordance for amplicons generated from 0.5 ng with Wpa (99.88% and 99.69%) were also close to the technical replicates. In this case, the call rates of Wpa were slightlyreduced compared to that with 5 ng input, but the call rate for the partially degraded sample 04-018, was modestly improved over Gv2 (92.06 % vs. 90.53%). Wpa data also showed some amplification non-uniformity among different locations, resulting in some âartificial CNVsâ similar to Gv2 (exampled as in Suppl. Fig. 5 and Suppl. Table 6), with the outputs obtained by taking unamplified gDNAs as their reference. This imbalance however was consistent and reproducible for each method but different between Wpa and Gv2. These artificial CNVs can be efficiently cancelled if pair-wise amplified test and reference are compared, as observed in CGH result (Fig. 4 and Suppl. Fig. 4), also supported by others {Pugh 2008}. It is interesting to note that the representation of chromosomal terminal sequences was greatly improved with Wpa compared with Gv2 (Fig. 5), and that some of these regions were significantly under-amplified or even lost with Gv2 (Suppl. Fig. 5 and Suppl. Table 6, 7), as also independently reported recently {Pugh 2008}. This occurred especially in the terminal 3 to 5 Mb and sometimes extended to 10 Mb in many chromosome termini, and was particularly serious when low levels or degraded DNA was as input. An analysis for 5 Mb termini is shown (Suppl. Table 5b calculated all involved SNPs as a cohort. Fig. 5 and Suppl. Tables 6 and 7 were the result for each chromosome terminus). Importantly, the SNP typing was also greatly improved, outstandingly exemplified by the amplicons of 0.5 ng input for the partially degraded 04-018, with Wpa versus Gv2 call rate of 91.9% vs. 84.45% and accuracy of 99.57% vs. 98.62%. The result also showed that these terminal regions underrepresentation in Gv2 was not absolutely associated with the distance-to-end, but possibly was a sequence related issue. Keywords: Whole-pool amplification, whole genome SNP typing The overall goal of the part of study was a validation of the quality of the amplicons from different amounts (5ng and 0.5 ng) of original starting gDNA, good quality (sample 05-050) or partially degraded gDNA (sample 04-018), with our new procedure Wpa, and with native gDNA as control, in terms of the call rate and accuracy (allele bias) in addition to the uniformity of the sequence amplified (sequence representation or sequence bias). Amplified or native genomic DNA isolated from patients was in-parallel analyzed/genotyped with the same experimental platform, of which the native genomic DNAs were used as the standard controls. For the sequence representation, the two alleles of the SNPsâ signal of a panel of multiple native DNAsâ signal provided by the experimental platform (Illumina) was used as the reference, so that an abstract signal for sequence representation of each SNP and for all SNPs was obtained.

Dataset Information

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized U87MG or eHAP cells after live/dead selection with Reovirus Type 3 Dearing Strain.

Publications

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

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