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| Status |
Public on Apr 16, 2019 |
| Title |
HE_cropseq_rep1 |
| Sample type |
SRA |
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| Source name |
Hepatic endoderm
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| Organism |
Homo sapiens |
| Characteristics |
cell_line: H1
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| Treatment protocol |
For perturbation experiments, H1-AAVS1-TetOn-dCas9-KRAB hESCs were transduced with individual gRNA lentiviral constructs and selected with puromycin for 72 hours. 48 hours prior to the start of differentiation, the selected cells were pooled and treated with 500 ng/mL doxycycline. The cells were then split for differentiation and differentiated along published protocols in the presence of 500 ng/mL doxycycline.
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| Growth protocol |
H1-AAVS1-TetOn-dCas9-KRAB hESCs were maintained in mTeSR1 on hES qualified matrigel-coated plates
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Drop-Seq Laboratory Protocol v3.1, Macosko et al. Cell 2015 (hepatic endoderm)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
polyadenylated RNA
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| Data processing |
ScreenProcessing (https://github.com/mhorlbeck/ScreenProcessing) was used to quantify guide abundance in genomic DNA.
QuantSeq reads were aligned using hisat2 v2.0.5 with hg19 as a reference and parameters “-p 12 --rna-strandness F”. Quantification used ESAT v0.1 with parameters “-wLen 100 -wOlap 50 -wExt 1000 -sigTest .01 -multimap normal”
10X Genomics Cellranger version 2.1.0 was used for fastq generation, alignment, and quantification of 10X data (definitive endoderm). The reference FASTA and GTF were prepended with customized sequences and annotations (attached as supplemental files) to facilitate alignment and tagging of guide RNAs and dCas9-KRAB.
Drop-seq tools version 1.0 was used for alignment and quantification of drop-seq data (hepatic endoderm).
Alignment and quantification were performed using the STAR aligner v2.4.2, Picard tools v1.96, Samtools v1.3, and Drop-seq tools v1.0. Details follow.
Note: fastq (R1 is a barcode)
Analysis step: Convert to SAM Software package: Picard tools Command name: FastqToSam.jar Parameters used: --
Analysis step: Tag cell barcode Software package: DropSeq tools Command name: TagBamWithReadSequenceExtended Parameters used: BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1, DISCARD_READ=False
Analysis step: Tag molecular barcode Software package: DropSeq tools Command name: TagBamWithReadSequenceExtended Parameters used: BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1, DISCARD_READ=True
Analysis step: Remove low-quality barcodes Software package: DropSeq tools Command name: FilterBAM Parameters used: --
Analysis step: Remove polyA tail Software package: DropSeq tools Command name: PolyATrimmer Parameters used: MISMATCHES=0, NUM_BASES=6
Analysis step: Convert to FASTQ Software package: Picard tools Command name: SamToFastq.jar Parameters used: --
Analysis step: Align reads to exome Software package: STAR Command name: -- Parameters used: --
Analysis step: Sort BAM to speed merging Software package: Picard tools Command name: SortSam.jar Parameters used: --
Analysis step: Merge barcode and alignment info Software package: Picard tools Command name: MergeBamAlignment.jar Parameters used: INCLUDE_SECONDARY_ALIGNMENTS=False
Analysis step: Label read with exon Software package: DropSeq tools Command name: TagReadWithGeneExon Parameters used: --
Analysis step: Screen for bead errors Software package: DropSeq tools Command name: DetectBeadSynthesisErrors Parameters used: NUM_BARCODES=2000
Analysis step: Form DGE matrix Software package: DropSeq tools Command name: DigitalExpression Parameters used: MIN_NUM_GENES_PER_CELL=1000
Genome_build: hg19 plus guide RNA and dCas9-KRAB sequences
Supplementary_files_format_and_content: Processed files are digital gene expression matrices containing UMI counts. For Drop-seq data (hepatic endoderm), they are represented as dense matrices in (gzipped) tab-delimited text files. Each column is labeled with a cell barcode and each row is labeled with a gene. For 10X data (definitive endoderm), they are represented as gzipped TAR archives containing MEX-format digital gene expression matrices.
Supplementary_files_format_and_content: DE_TERA_rep1.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep2.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep1_gAmp.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep2_gAmp.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: star_perturbseq_HE_rep1_merged_gene_exon_tagged_clean.dge.txt: Dense digital gene expression matrix (gzipped tab-separated text)
Supplementary_files_format_and_content: star_perturbseq_HE_rep2_merged_gene_exon_tagged_clean.dge.txt: Dense digital gene expression matrix (gzipped tab-separated text)
Supplementary_files_format_and_content: DE1_gDNA_ryan_guides_2018JAN19_unique_only.fa.counts: Whitespace-separated two-column text file; guide names followed by counts.
Supplementary_files_format_and_content: DE2_gDNA_ryan_guides_2018JAN19_unique_only.fa.counts: Whitespace-separated two-column text file; guide names followed by counts.
Supplementary_files_format_and_content: quantseq_timecourse.quant.gene.txt: Typical ESAT output: whitespace-separated text file with header containing sample names and body containing counts. First three columns contain gene symbol, chromosome, and strand.
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| Submission date |
Feb 26, 2019 |
| Last update date |
Apr 17, 2019 |
| Contact name |
Rene Maehr |
| Organization name |
UMass Medical School
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| Department |
Program in Molecular Medicine
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| Lab |
Diabetes Center of Excellence
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| Street address |
55 Lake Ave N
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01604 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE127199 |
Single-cell RNA sequencing-based CRISPRi screening resolves molecular drivers of early human endoderm development [set 1] |
| GSE127202 |
Single-cell RNA sequencing-based CRISPRi screening resolves molecular drivers of early human endoderm development |
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| Relations |
| BioSample |
SAMN11022756 |
| SRA |
SRX5431783 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3630204_star_perturbseq_HE_rep1_merged_gene_exon_tagged_clean.dge.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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