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| Status |
Public on Mar 06, 2015 |
| Title |
Replicate 2, RNA-Seq, time 0 |
| Sample type |
SRA |
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| Source name |
bone marrow-derived primary dendritic cells
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| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: C57BL/6J
treatment: mock-stimulated
time point: 0
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| Treatment protocol |
At day 9, cells were stimulated for various time points with LPS (100ng/ml, rough, ultrapure E. coli K12 strain, Invitrogen) or MOCK (= no stimulation).
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| Growth protocol |
6-8 week old female C57BL/6J mice were obtained from the Jackson Laboratories. RPMI medium (Invitrogen) supplemented with 10% heat inactivated FBS (Invitrogen), ß-mercaptoethanol (50uM, Invitrogen), L-glutamine (2mM, VWR), penicillin/streptomycin (100U/ml, VWR), MEM non-essential amino acids (1X, VWR), HEPES (10mM, VWR), sodium pyruvate (1mM, VWR), and GM-CSF (20 ng/ml; Peprotech) was used throughout the study. At day 0, bone marrow-derived dendritic cells (BMDCs) were collected from femora and tibiae and plated on twenty (per mouse), 100mm non tissue culture treated plastic dishes using 10ml medium per plate. At day 2, cells were fed with another 10ml medium per dish. At day 5, cells were harvested from 15ml of the supernatant by spinning at 1,400 rpm for 5 minutes; pellets were resuspended with 5ml medium and added back to the original dish. Cells were fed with another 5ml medium at day 7. At day 8, all non-adherent and loosely bound cells were collected and harvested by centrifugation. Cells were then resuspended with medium, plated at a concentration of 10x106 cells in 10ml medium per 100mm dish.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells using RNeasy Mini Kit (Quiagen), according to the manufacturer’s protocol.
Enrichment of polyadenylated RNA (polyA+ RNA) from total RNA was performed using Oligo(dT) dynabeads (Invitrogen) according to the manufacturer’s protocol. The mRNA was chemically fragmented into ~80-nt-long fragments using RNA fragmentation reagent (Ambion), followed by Turbo DNAse treatment (Ambion). Strand-specific RNA-seq libraries were generated as previously described (Engreitz et al., Science, 2013). Briefly, RNA was first subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (Agilent), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9- to 14 cycles in a PCR reaction. Libraries were sequenced on an Illumina HiSeq 2500 generating 30bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
processed data column name: RNA.R2.MOCK.00 aka RNA.R2.LPS.00
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| Data processing |
We constructed “analysis groups” using the following rules: 1. All transcripts in the same transcript group are in the same analysis group, where transcript groups are defined by the UCSC Table Browser (query settings: assembly=”July 2007 (NCBI37/mm9)”, group=”Gene and Gene Predictions”, track=”UCSC Genes”, table=”knownIsoforms”; see the fields “clusterID” and “transcript”) and represent groups of transcripts (isoforms) that are derived from the same genic locus and cannot easily be resolved. 2. All proteins in the same MaxQuant protein group are in the same analysis group, where MaxQuant determines protein groups on the basis of the peptide library supplied. The groups represent the level of ambiguity MaxQuant can confidently resolve. 3. If a protein and a transcript are associated with one another, then they are in the same analysis group, where we associate (one or more) Uniprot protein ID with (one or more) UCSC transcript ID based on ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/idmapping/by_ organism/MOUSE_10090_idmapping.dat
We created a Bowtie15 index based on annotated transcripts from UCSC mm9 and aligned paired-end reads directly to this index using Bowtie v 0.12.7 with command line options: -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -a –m 200. Next, we ran RSEM v1.1117 (with default parameters) and used the analysis groups (described above) to define which isoforms belong to the same gene. Analysis groups with no associated proteins (i.e. noncoding RNAs) were still used as input to avoid incorrect mappings. Expression quantifications were made for each analysis group, replicate, and condition.
Analysis groups (henceforth referred to as "genes" for simplicity) were excluded if they were not MS2-identified in at least 6 of the 10 time points in all conditions (time courses for LPS and MOCK stimulation), channels, and replicates. After applying this filter, we also excluded genes that did not have positive RNA-Seq values for at least 6 out of the 10 time points in all conditions and channels. We renormalized such that at each condition/time point the RNA-Seq expression values added up to exactly 1,000,000. Meanwhile, we normalized the protein data such that at each condition/time point protein (M+H) IBAQ values added up to exactly 1,000,000 (missing values were treated as zeroes).
For the shRNA RNA-Seq data: We created a Bowtie index based on the UCSC knownGene transcriptome, and aligned paired-end reads directly to this index using Bowtie v0.12.7 with command line options —-norc q --phred33-quals -n 2 -e99999999 -l 25 -I 1 -X 1000 -a -m 200, reflecting our strand-specific data. The output of this reflects the transcriptome alignment percentage for each library, which ranged between 57-65%. To calculate expression levels, we used this alignment as input for RSEM (v. 1.2.3) with default parameters as well as the -- forward-prob 1 and estimate-rspd options. We used the TPM output of RSEM as a normalized estimate of gene expression for each sample.
Genome_build: mm9
Supplementary_files_format_and_content: protein.norm.csv: normalized M-channel and H-channel protein expression values for each replicate, time point, and experimental condition
Supplementary_files_format_and_content: rna.norm.csv: normalized RNA expression values for each replicate, time point, and experimental condition
Supplementary_files_format_and_content: shRNA_RNAseq.xlsx: RNA-Seq expression (TPM) of genes according to respective knock down
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| Submission date |
Jul 25, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Catherine J Wu |
| Organization name |
Dana Farber Cancer Institute
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| Lab |
Wu Lab
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| Street address |
450 Brookline Ave.
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE59784 |
Dynamic profiling of the protein life cycle in response to pathogens (RNA-seq) |
| GSE59793 |
Dynamic profiling of the protein life cycle in response to pathogens |
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| Relations |
| BioSample |
SAMN02941090 |
| SRA |
SRX661675 |
| 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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