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Sample GSM1315486 Query DataSets for GSM1315486
Status Public on Mar 02, 2014
Title Microglia-RNAseq-wt
Sample type SRA
Source name Primary Microglia (wt HTT Q7/7)
Organism Mus musculus
Characteristics cell type: microglial
htt status: wildtype HTT Q7/7
Treatment protocol pCDH, Nwt, and Nmu Constructs: N-terminus human wild-type and N-terminus mutant huntingtin have been cloned from pCAG-Htt19550-15Q and pCAG-Htt1955-128Q respectively into MCS of pCDH-CMV-MCS-EF1-Puro (System Bioscience) using EcoRI and NotI. Lentiviral production and BV2 cells transduction were performed according to the manufacturer's protocol. Control cell line was generated by transducing BV2 cells with Lentivirus obtained from pCDH-CMV-MCS-EF1-Puro (empty vector). Validation of plasmids used in this study was performed by Western blotting.
Growth protocol Murine microglial BV2 cells, primary mouse microglia, mouse astrocyte maintained with DMEM (Cellgro) supplemented with 10% FBS (low endotoxin, Hyclone) and penicillin/streptomycin (Invitrogen). Adult microglia Purification: Cortex, striatum, and hippocampus were extracted and gently homogenized in staining buffer (HBSS (Life Technologies, 14175-095), 1% BSA, 1mM EDTA) on ice using a 2 ml polytetrafluoroethylene pestle (Wheaton), first in a 14 ml round-bottom tube (BD Falcon, 352059) and then in a 2 ml grinder chamber (Wheaton, 358029). Homogenates were filtered onto a 70 ?m cell strainer (BD Falcon, 352350) and centrifuged for 10 min at 400g. Cell pellets were resuspended in 6 ml of 37% isotonic Percoll (Sigma, P4937) and then underlayed with 5 ml of 70% isotonic Percoll in a 15ml centrifuge tube (Corning, 430790). Tubes were then centrifuged at 600g for 40 min at 18°C, with no acceleration or decelaration. Cells at the 37?70% Percoll interface were recovered and washed once in 15 ml HBSS. Cell were then incubated in staining buffer on ice with CD16/CD32 (eBioscience, clone 93) antibody for 25 min, and then with CD11b-PE (BioLegend 101208, clone M1/70) and CD45- Alexa488 (BioLegend 103122, clone 3-F11) antibodies for 30 min. Cells were then washed once and filtered onto a 40 ?m cell strainer (BD Falcon, 352340). Sorting was performed on a BD Influx cell sorter. Microglia were defined as singlets, CD11b+CD45Low events, and emcompassed 90-95% of all CD11b+ events. BMDM Isolation: Bone marrow was harvested and washed once with PBS, seeded/cultured in DMEM containing 20 % FCS, 30 % L-cell conditioned media (as source of macrophage colony stimulating factor, M-CSF), and 100 U/ml penicillin/streptomycin in non-tissue culture-treated petri dishes. After 6-8 days of culture, non-adherent cells were washed off with room temperature PBS and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in RPMI 1640 containing 10% FCS, 100 U/ml penicillin/streptomycin, and 10ng/ml M-CSF.
Extracted molecule polyA RNA
Extraction protocol RNA was purified using RNeasy Mini Kit (Qiagen) and enriched for Poly(A)-RNA with MicroPoly(A) Purist Kit (Ambion). Subsequently, RNA was treated with TURBO DNase (Ambion), fragmented with RNA Fragmentation Reagents (Ambion) and purified by a P-30 column (Biorad).
Fragmented RNA was dephosphorylated with Antarctic phosphatase (New England Biolabs) heat inactivated and precipitated over-night. Poly(A)-tailing and cDNA synthesis was performed as previously described. For reverse transcription, oligos with custom barcodes (underlined) were used: 5′-Phos-CA/TG/AC/GT GATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGATTTT TTTTTTTTTTTTTTTTVN-3′. Subsequently, exonuclease was used to remove the excess oligo. After heat-inactivation, RNA was hydrolyzed by alkaline treatment (100mM NaOH) and heat at 95°C for 25 min. The cDNA fragments of 50–150 nucleotides were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 12 cycles, and gel purified as previously described. The library was sequenced on Illumina sequencers according to the manufacturer's instructions.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description no treatment
Data processing RNA-Seq: Reads were aligned to the mouse mm9 genome (NCBI Build 37) using STAR (v2.0.3e). RNA-Seq experiments were normalized and visualized by using HOMER ( to generate custom tracks for the UCSC Genome Browser ( Gene expression values were generating for RefSeq annotated transcripts using HOMER and differential expression calculations were performed using edgeR.
RNA-Seq RPKM tab-delimited text file is available on series record. Columes: 1: RefSeq ID, 2: chr, 3: gene start, 4: gene end, 5: strand, 6: mRNA length, 7: Copies of gene in genome, 8: Annotation information, 9-18: RPKM gene expression values
Genome_build: mm9
Submission date Jan 27, 2014
Last update date May 15, 2019
Contact name Christopher Benner
Organization name University of California, San Diego (UCSD)
Department Medicine
Street address 9500 Gilman Dr. MC 0640
City La Jolla
State/province California
ZIP/Postal code 92093-0640
Country USA
Platform ID GPL13112
Series (1)
GSE54443 Mutant Huntingtin promotes neuronal death through cell autonomous microglial activation via myeloid lineage- determining factors
Reanalyzed by GSE80797
BioSample SAMN02598193
SRA SRX451628

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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