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| Status |
Public on Jun 18, 2019 |
| Title |
Donor HU1007 (AKA Sample 1) uninfected, 8 days |
| Sample type |
SRA |
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| Source name |
SACC-PHHs
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: Primary human hepatocytes co-cultured with mouse 3T3J non-parenchymal cells
treatment: Uninfected
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| Treatment protocol |
PHHs (188,000 cells/well) were seeded along with 3T3 J mouse (30,000 cells/well) in a 24 well format. Seven to ten days after seeding, SACC-PHHs were challenged with respective viruses.
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| Growth protocol |
All SACC-PHHs were grown in Hurel Maintenance media containing 5% fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen Rneasy Mini Kit per manufacturer's directions.
The integrity of total RNA samples were assessed on Bioanalyzer 2100 using RNA 6000 Nano chip (Agilent Technologies, CA); only samples with RNA Integrity Number (RIN) greater than 9.0 were used for RNA-seq. The poly-A containing RNA transcripts were enriched from 1μg of total RNA for each sample using oligo-dT bead, and further converted to cDNA then Illumina sequencing library using PrepX RNA-seq library kit on the automated Apollo 324TM NGS Library Prep System (Wafergen Biosystems, CA) according to the manufacturer's protocol. Different DNA barcodes were added to each RNA-seq library. The libraries were examined on Agilent Bioanalyzer DNA High Sensitivity chips for size distribution, and quantified by Qubit fluorometer (Invitrogen, CA). The RNA-seq libraries were pooled at equal molar amount and sequenced on Illumina HiSeq 2500 Rapid flowcells as single-end 75 nucleotide reads following the standard protocol. Raw sequencing reads were filtered by Illumina HiSeq Control Software, and only the Pass-Filter (PF) reads were used for further analysis.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
35_Ctrl_D8_sample_1
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| Data processing |
The samples were run on two HiSeq 2500 Rapid Run mode flowcells. Basecalling was performed by Illumina RTA version 1.18.64.0. BCL files were then converted to fastq format using Illumina’s bcl2fastq version 1.8.4. Reads that aligned to phiX using Bowtie 1.1.1 (Langmead et al., 2009) were removed as well as reads that failed Illumina’s default chastity filter. The fastq files from each lane were then combined before splitting the reads from each sample using the barcode sequences allowing 1 mismatch, using the barcode_splitter program, version 0.18.2 (Leach and Parsons, 2017).
A reference genome was created using the NCBI GRCh38 human reference (iGenomes), the NCBI GRCm38 mouse reference (iGenomes), the NCBI U95551.1 Hepatitis B virus subtype ayw, complete genome, and the NCBI M21012.1 Hepatitis delta virus RNA, complete genome. The reference sequences (fasta) and gene annotations (gtf) were combined to form a single unified reference. The read alignment, quantification, and differential expression analysis was done using a locally installed instance of the Galaxy workflow platform (Afgan et al., 2016). The reads for each sample were then aligned to the combined reference using STAR (Galaxy Version 2.5.2b-1) with default parameters (Dobin et al., 2013). Quantification for each gene was then done using featurecounts (Galaxy Version 1.6.0.1) with default settings (Liao et al., 2014).
Genome_build: A reference genome was created using the NCBI GRCh38 human reference (iGenomes), the NCBI GRCm38 mouse reference (iGenomes), the NCBI U95551.1 Hepatitis B virus subtype ayw, complete genome, and the NCBI M21012.1 Hepatitis delta virus RNA, complete genome.
Supplementary_files_format_and_content: Tabular files of ENSEMBL gene ID and counts
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| Submission date |
Mar 20, 2018 |
| Last update date |
Jun 20, 2019 |
| Contact name |
Alexander Ploss |
| E-mail(s) |
aploss@princeton.edu
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| Phone |
609-258-7128
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| Organization name |
Princeton University
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| Department |
Molecular Biology
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| Lab |
110 Lewis Thomas Laboratory
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| Street address |
Washington Road
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| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
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| Platform ID |
GPL22245 |
| Series (1) |
| GSE112118 |
RNASeq of total RNA isolated from self-assembling co-cultures of primary human hepatocytes (SACC-PHHs) mono-infected with HBV, co-infected with HBV/HDV, or uninfected at 8 and 28 days post-infection |
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| Relations |
| BioSample |
SAMN08744676 |
| SRA |
SRX3825843 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3058088_Ctrl_D8_sample_1.tabular.txt.gz |
444.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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