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Sample GSM1131274

Query DataSets for GSM1131274

Status

Public on Oct 15, 2013

Title

RNA-Seq BALBc/J no treatment

Sample type

SRA

Source name

peritoneal thioglycolate-elicited macrophages

Organism

Mus musculus

Characteristics

strain: BALBc/J
treatment: no treatment
chip (antibody): N/A

Treatment protocol

Cells were treated by replacing the seeding media with fresh, warm media ("notx") or fresh, warm media containing 100 ng/ml KDO2-lipid A (KLA) for the indicated times.

Growth protocol

Peritoneal macrophages were harvested by peritoneal lavage with 20 ml ice-cold PBS 4 days after peritoneal injection of 3 ml thioglycollate broth. Peritoneal cells seeded in 10% fetal calf serum (FCS)/RPMI-1640 containing 100 U/ml penicillin/streptomycin in tissue culture-treated petri dishes overnight. Non-adherent cells were washed off with room temperature PBS.

Extracted molecule

polyA RNA

Extraction protocol

RNA-Seq: PolyA-selected RNA (MicroPoly(A)Purist kit (Ambion)) was hydrolyzed with Fragmentation Buffer (Ambion) for 10 min at 70°C and re-buffered to 10 mM Tris pH 7.4 using P-30 size exclusion columns (Bio-Rad). Thirty nanograms RNA were de-capped for 2 h at 37°C with 0.5 µl (10 U/µl) TAP (Epicentre) in 20 µl TAP buffer containing 1 U/µl SUPERase-IN. Samples were 3’ dephosphorylated for 50’ at 37°C with 1 µl PNK (Enzymatics), 0.5 µl 10x TAP buffer, 1.5 µl water, 0.5 μl 0.25 M MgCl2 (3 mM free Mg2+ final), 0.5 μl 10 mM ATP (0.2 μM final to protect PNK). Subsequently, RNA was 5’-phosphorylated for 60 min at 37°C by adding 2 μl (10 U/µl) PNK, 10 μl 10x T4 DNA ligase buffer and 63 μl water. RNA was extracted with Trizol LS, precipitated in the presence of Glycoblue (Ambion), and dissolved in 4.5 µl water. 0.5 µl 9 µM of a 5’-adenylated sRNA3'MPX adapter /5Phos/AG ATC GGA AGA GCA CAC GTC TGA /3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer’s instructions, phenol-chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 µM) were heat-denatured together with the RNA for 2 minutes at 70°C, and ligated with 100 U truncated T4RNA ligase 2 K227Q (NEB) in 10 µl 1x T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hours at 16°C. To reduce adapter dimer formation, 0.5 μl 10 μM MPX_ RT primer 5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 minutes, then 37°C for 30 minutes, then 25°C for 15 minutes. Finally, 0.5 µl 5 µM hybrid DNA/RNA sRNA5'h adapter 5’-GTT CAG AGT TCT ACA rGrUrC rCrGrA rCrGrA rUrC-3’ (IDT) were ligated to previously capped RNA 5’ ends by adding 2 µl T4 RNA ligase buffer, 6 µl 50% PEG8000 (15% final), 1 µl 10 mM ATP, 9.5 µl water and 0.5 µl (5 U) T4 RNA ligase 1 for 90 minutes at 20°C. To 15 μl ligation reaction, an additional 0.5 μl 10 μM MPX_ RT primer were added, reactions were denatured at to 70°C for 1 minute, then placed on ice. RNA was reverse-transcribed by adding 3 µl 10x first strand buffer, 4.5 µl water, 1.5 µl 10 mM dNTP, 3 µl 0.1 M DTT, 1.5 µl RNaseOUT and 1 µl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. Complementary DNA was isolated by adding 35 µl AMPure XL beads (Beckman), binding and washing according to manufacturer’s instructions and dissolved in 40 µl TET. Libraries were PCR-amplified for 11 cycles with 0.75 µM primers oNTI201 (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAC G-3' and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT iii iii GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, i signifies index nucleotides) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ∞), 175-225 bp fragments were size-selected on 10% PAGE gels and sequenced for 51 cycles on a HiSeq 2000 sequencer (Illumina) with small RNA sequencing primer 5’-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3’ and TruSeq Index sequencing primer (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

GeneExpression.txt

Data processing

For ChIP-Seq, RNA-Seq and GRO-Seq samples, reads were aligned to the mm9 genome as well as BALB/cJ contigs using bowtie (v0.12.7), keeping only reads that mapped best to a single, unique location in both genomes, while allowing 3 mismatches in the first 28 bases, and at least 5 high-quality mismatches overall. The NOD PU.1 notx ChIP-Seq sample was only mapped to mm9 according to the same criteria. To identify allele-specific reads in CB6F1J, the initial mapping to strain-specific regions in both genomes defined in the parental inbred strains was performed with the leftmost 33 bases allowing no mismatch, and reads that mapped to both genomes were discarded. Aligned read files were analyzed with HOMER (http://biowhat.ucsd.edu/homer/) to find peaks, calculate RPKM from the gene bodies of RefSeq genes, perform motif- and other analyses in the study. Genetic variation analyses were performed using custom R scripts.
Genome_build: mm9, BALB/cJ de novo assemblies (Keane, T. M. et al., Nature 477, 289-294 (2011), the initial published version) were downloaded from the Sanger Institute website: http://www.sanger.ac.uk/resources/mouse/genomes/
Supplementary_files_format_and_content: Processed files include BED-like files (ChIP-Seq peak positions), rpkm text files (RNA-Seq, GRO-Seq expression data). All genomic coordinates are relative to mm9 (NCBI 37) mouse assembly.

Submission date

Apr 30, 2013

Last update date

May 15, 2019

Contact name

Casey Romanoski

Organization name

UCSD

Street address

9500 Gliman Dr.

City

La Jolla