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Sample GSM1155643 Query DataSets for GSM1155643
Status Public on Feb 12, 2014
Title 1119_SP
Sample type SRA
Source name Serrated polyp tissue from HBUS mouse 1119
Organism Mus musculus
Characteristics strain: HBUS
tissue: Serrated polyp
Growth protocol HBUS mice were reared as described (Bongers et al., 2010; Bongers et al., 2012) and cecal polyp/surrounding tissue was isolated after 60 weeks. All experiments involving mice were performed in accordance with the guidelines of the Animal Care and Use Committee of Mount Sinai School of Medicine.
Extracted molecule total RNA
Extraction protocol Approximately 200 mg tissue sample was placed in 1.5 ml RNAlater buffer (Ambion) and snap-frozen in liquid nitrogen. At time of extraction, samples were centrifuged for 10 min at 16.000g at room temperature. Pellets were resuspended in 150 μl of Lysis Buffer (Tris/HCl pH 8, 1 mM EDTA, 15 mg/mL Lysozyme (Sigma), 15 μl Proteinase K (20 mg/mL)) and incubated at room temperature for 10 min with brief mixing every 2 min. Following addition of 1.2 mL QIAGEN RLT buffer containing 1% v/v β-mercaptoethanol, 1 mL of 0.1 mm glass beads (BioSpec) were added and samples were homogenized in a FastPrep at setting 5 (4 pulses of 20 sec). Samples were kept on ice for 1 min between pulses. Lysates were then homogenized with a QIAshredder spin columns and RNA was isolated using the AllPrep mini kit (Qiagen), according to the manufacturers protocols, which included an on-column digestion with DNase I. 25 μg of RNA from each tissue sample was processed with the MICROBEnrich kit (Ambion) and 5 μg of processed RNA was further depleted of rRNAs using the Meta-Bacteria RiboZero rRNA removal kit (Epicentre).
rRNA depleted RNA was prepared for Illumina paired-end sequencing using the NEB Next mRNA Library Prep Master Mix Set for Illumina (NEB, E6110S); manufacturer’s protocols were followed with the following modifications. RNA was fragmented for 10 minutes, and then purified with Qiagen RNeasy MinElute spin columns. Reverse transcription was performed with SuperScript III (Invitrogen). Library preparation reactions were cleaned up using Agencourt AMPure XP beads. Size selection was performed before ligation mediated PCR using Invitrogen E-Gel 2% with SYBR Safe staining. Excised gel fragments were purified with Qiagen QIAQuick Gel Extraction kit. Adapters and primers were synthesized by IDT following published Illumina sequences. Enrichment PCR was performed with Kapa HiFi HotStart ReadMix. Primers were used at a final concentration of 500 nM; cycling parameters were as follows: 94 oC for 5 min, 15 cycles of 94 oC 1 min, 62 oC for 30 sec, 72 for 45 sec and then 72 oC for 10 min. Libraries were quantified using the Agilent BioAnalyzer DNA 1000 chips, diluted to 12 pM and sequenced for 100 cycles (paired-end) on the Illumina HiSeq 2000 using standard methods.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description Serrated polyp tissue from HBUS mouse 1119
Data processing Paired-end sequencing reads were aligned to the M. musculus genome using Tophat (v2.0.7) with the –bowtie1 argument (bowtie v0.12.9). Read counts were extracted using htseq-count (v0.5.3p9).
Genome_build: MGSCv37
Supplementary_files_format_and_content: Read counts extracted using htseq-count (v0.5.3p9).
Submission date Jun 07, 2013
Last update date May 15, 2019
Contact name Gerold Bongers
Organization name Icahn School of Medicine at Mount Sinai
Department Immunology Institute
Street address 1425 Madison Ave, 12-26A
City New York
State/province NY
ZIP/Postal code 10029-6574
Country USA
Platform ID GPL13112
Series (2)
GSE47735 Interplay of host microbiota, genetic perturbations, and inflammation promotes local development of intestinal neoplasms in mice [RNA-Seq]
GSE47736 Interplay of host microbiota, genetic perturbations, and inflammation promotes local development of intestinal neoplasms in mice
BioSample SAMN02192692
SRA SRX297978

Supplementary file Size Download File type/resource
GSM1155643_1119_SP_raw-gene-counts.txt.gz 131.1 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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