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| Status |
Public on Nov 27, 2017 |
| Title |
Sample_54968 |
| Sample type |
SRA |
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| Source name |
Isolated pancreatic acinar cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: PDX1f/f;Ptf1aCreERT
cell type: Isolated pancreatic acinar cells
treatment: TGFa Day 2
molecule subtype: Ribo depleted RNA
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| Treatment protocol |
Pancreata were harvested, washed twice in HBSS, minced, and incubated with 0.2 mg/mL Collagenase P (Roche;11249002001) at 37 degrees C for 15 minutes. Tissue was washed 3 times in HBSS containing 5% FBS and filtered through 500-um and 105-um polypropylene mesh (Spectrum Laboratories;106436 and 106418). Filtrate was centrifuged through 30% FBS in HBSS and resuspended in complete Waymouth media (1x Waymouth MB 752/1 medium, 50 mg/mL gentamycin, 0.4 mg/mL soybean trypsin inhibitor, and 1 mg/mL dexamethasone). For RNAseq experiments, suspension was plated in non-adherent plates, treated with TGF? (50 ng/mL) and maintained at 370C in a 5% CO2 atmosphere for 1-3 days.
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| Growth protocol |
To induce acinar specific recombination, Ptf1a-CreERT, or Pdx1f/f;Ptf1aCreERT mice were administered tamoxifen (Sigma T5648) P.O. once daily for five days at 5 mg per 20g of body weight.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using the RNEasy-Plus Kit (Qiagen;74136) and assessed for quality using the TapeStation (Agilent). Samples with RNA Integrity Number of 8 or greater were rRNA depleted using Ribo Minus (Invitrogen;K1550-04).
rRNA-depleted samples were prepared using the TruSeq mRNA SamplePrep v2 kit (Illumina;RS-122-2001, RS-122-2002). The entire fraction of 0.1-3 ug of rRNA depleted total RNA was fragmented and copied into first-strand cDNA using reverse transcriptase and random primers. cDNA 3'-ends were adenylated and adapters were ligated. One of the adapters had a 6-nucleotide barcode that was unique for each sample, allowing us to multiplex in a HiSeq flow cell (Illumina). Products were purified and enriched by PCR to create the final cDNA library. Libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using a library quantification kit for Illumina platforms (Kapa Biosystems;KK4835). They were clustered on the cBot (illumina) and sequenced 24 samples per lane on 6 lanes of a 50-cycle single end HiSeq 4000. HiSeq Control Software version 3.3.52 was used according to manufacturer?s protocols. Demultiplexing and Fastq file generation was done using bcl2fastq version 2.17.1.14.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
KO_Day_2_1
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| Data processing |
QC: We checked the quality of the raw reads data for each sample using FastQC [1] (version 0.10.1) to identify features of the data that may indicate quality problems (e.g. low quality scores, over-represented sequences, inappropriate GC content, etc.).
Aligment: We used the software package Tuxedo Suite for alignment, differential expression analysis, and post-analysis diagnostics [2, 3, 4]. Briefly, we aligned reads to the reference transcriptome including both mRNAs and lncRNSs (UCSC mm10) [5] using TopHat (version 2.0.9) and Bowtie (version 2.1.0.). We used default parameter settings for alignment, with the exception of: “--b2-very-sensitive” telling the software to spend extra time searching for valid alignments.
QC: We used FastQC for a second round of quality control (post-alignment), to ensure that only high quality data would be input to expression quantitation and differential expression analysis.
Normalization and Quantitation: We used Cufflinks/CuffDiff (version 2.1.1) for expression quantitation, normalization, and differential expression analysis, using UCSC mm10.fa as the reference genome sequence. For this analysis, we used parameter settings: “--multi-read-correct” to adjust expression calculations for reads that map in more than one locus, as well as “--compatible-hits-norm” and “--upper-quartile –norm” for normalization of expression values. We generated diagnostic plots using the CummeRbund package.
Differential Expression: We used locally developed scripts to format and annotate the differential expression data output from CuffDiff. Briefly, we identified genes and transcripts as being differentially expressed based on three criteria: test status = “OK”, FDR < 0.05 and fold change ≥ ± 1.5.
Annotation: We annotated genes and isoforms with NCBI Entrez GeneIDs and text descriptions. We further annotated differentially expressed genes with Gene Ontology (GO) [6] terms using NCBI annotation.
Enrichment Testing: We used DAVID (version 6.7) [7] for enrichment analysis of the set of differentially expressed genes to identify significantly enriched functional categories.
Genome_build: MM10
Supplementary_files_format_and_content: .xls table of normalized expression values for each gene for each sample
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| Submission date |
Dec 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard C McEachin |
| E-mail(s) |
mceachin@umich.edu
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| Organization name |
University of Michigan
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| Department |
DCM&B
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| Street address |
2800 Plymouth Road
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| City |
Ann Arbor |
| State/province |
United States |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE91052 |
PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [RNA-seq] |
| GSE91056 |
PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance |
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| Relations |
| BioSample |
SAMN06124617 |
| SRA |
SRX2405574 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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