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Sample GSM1554684 Query DataSets for GSM1554684
Status Public on Mar 06, 2015
Title Replicate 1, Ribosomal footprinting, resting DCs
Sample type SRA
Source name bone marrow-derived primary dendritic cells
Organism Mus musculus
Characteristics gender: female
strain: C57BL/6J
treatment: un-stimulated
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.
Extracted molecule total RNA
Extraction protocol Unstimulated BMDCs were incubated with 100µg/mL cycloheximide for 1 minute at 37°C. Cells were collected by centrifugation at 300 ×g for 5 minutes at 4°C and either washed twice with cycloheximide (100µg/mL) in ice-cold PBS or flash frozen in liquid nitrogen after removing media. Cell pellets were covered with 400µL of lysis buffer (4.75 mL polysome buffer + 250 µL 20% Triton X-100 (Sigma-Aldrich) + 60 units Turbo DNAse (Ambion)). Polysome buffer is 20mM Tris-HCl pH 7.56, 150mM NaCl, 5mM MgCl2, 100µg/mL CHX, 1mM DTT, and 8% glycerol. Lysis was carried out by triturating 10 times. Whole-cell lysates were clarified by centrifuging at 20,000 ×g for 10 minutes at 4°C. Clarified lysate was collected and flash frozen in liquid nitrogen.
Ribosome-protected fragments were isolated and cloned from lysates via RNAse I (Ambion) treatment as described previously (Ingolia et al., 2012). Samples were depleted of rRNA contamination with the Ribo-Zero rRNA Removal Kit (Epicentre) and sequenced on the Illumina HiSeq2500.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Data processing Sequencing reads were stripped of linker sequences using fastx_clipper from the FASTX-Toolkit with the following settings: -a CTGTAGGCACCATCAAT –n -Q 33. Reads mapping to miRNAs, rRNAs, snRNAs, snoRNAs, and tRNAs were filtered using bowtie2 in --local mode. Remaining reads were aligned using Tophat2 to the UCSC mm9 Known Gene transciptome. Tophat2 was run with the following settings --b2-very-sensitive --transcriptome-only --no-novel-juncs -max-multihits=64.
Footprints were filtered for read lengths 26-33 (inclusive). Footprints were mapped to their approximate P-site position by applying a 12 nucleotide offset from the 5' end. Footprint counts were averaged over the union of genomic positions corresponding to coding sequences of the genes in each “analysis group”, excluding positions within 3 nucleotides upstream or 15 nucleotides downstream of start codons or within 9 nucleotides upstream of stop codons.
The count of aligning nucleotides was divided by the length of the included region. This measure of ribosomal occupancy was renormalized to sum to 1 million across all genes.
Genome_build: mm9
Supplementary_files_format_and_content: ribosomalprofiling.csv
Submission date Nov 25, 2014
Last update date May 15, 2019
Contact name Catherine J Wu
Organization name Dana Farber Cancer Institute
Lab Wu Lab
Street address 450 Brookline Ave.
City Boston
State/province MA
ZIP/Postal code 02215
Country USA
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
BioSample SAMN03223274
SRA SRX769407

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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