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Sample GSM7851859

Query DataSets for GSM7851859

Status

Public on Jun 11, 2024

Title

Striatal Microglia, Saline, rep3

Sample type

SRA

Source name

Neuronal

Organism

Mus musculus

Characteristics

tissue: Neuronal
region: Striatum
cell type: Microglia
Sex: Male
treatment: Methamphetamine intravenous self administration
group: Saline

Treatment protocol

Male C57BL/6J mice (9 weeks old ~25-30 g; Jackson Laboratories, Bar Harbor, ME; SN: 000664) were housed in the animal facilities at the University of Miami Miller School of Medicine. Mice were maintained on a 12:12 h light/dark cycle (0600 hours lights on; 1800 hours lights off) and were housed 3 to 5 per cage. Animals were provided with food and water ad libitum. Mice representing each experimental group were evenly distributed among testing sessions. All animals were maintained according to the National Institutes of Health guidelines in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Miami Miller School of Medicine. Whenever possible, the experimenter was blind to the experimental and/or treatment group. For self-administration experiments in mice, methamphetamine hydrochloride (NIDA Drug Supply Program, Research Triangle Park, NC, USA) was dissolved in 0.9% sterile saline. Mice were permitted to self-administer methamphetamine or saline intravenous infusions during daily 2-hr sessions. Infusions were delivered through Tygon catheter tubing (Braintree Scientific, MA, USA) into the intravenous catheter by a variable speed syringe pump (Med Associates Inc, Fairfax, VT, USA). Self-administration sessions were carried out in operant chambers containing 2 retractable levers (1 active, 1 inactive) and a yellow cue light located above the active lever which illuminated during the intravenous infusion as well as during the post-infusion time out (TO). During Acquisition, mice started on a fixed ratio 1 (FR1) schedule of reinforcement for infusions 1 to 5, then FR2 for infusions 6 to 10, and FR3 for the remainder of the session. Completion of the response criteria on the active lever resulted in the delivery of an intravenous infusion (14 µL over 2 sec) of methamphetamine (0.05 mg/kg/infusion) or 0.9% saline. Responses on the inactive lever were recorded but had no scheduled consequences. After 5 consecutive days of Acquisition, mice were allowed to self-administer methamphetamine or saline during 10 consecutive daily 2-hr Maintenance sessions. Animals that did not demonstrate stable responding (less than 7 infusions per 2-hr session) or showed signs of compromised catheter patency were excluded from analysis. After 15 days of methamphetamine self-administration (Acquisition and Maintenance), mice underwent 21 days of forced home cage abstinence. Context-induced reinstatement session was then conducted on Day 21, where completion of response criteria resulted in the presentation of the light stimulus previously paired with methamphetamine or saline infusion delivery; however, no reward was delivered. Active lever presses were recorded and interpreted as a measure of drug-seeking.
Mice were anesthetized with isoflurane and perfused through the ascending aorta with 0.1 M phosphate buffer saline (PBS pH 7.4, Gibco, Waltham, MA, USA) plus heparin (7,500 USP units). Tissues were dissected and transported in Hibernate A Medium (Gibco) before dissociation. Briefly, tissue was enzymatically and mechanically dissociated, and debris removed using the Adult Brain Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The resulting single cell suspension was incubated with anti-mouse CD11b (i.e., microglia-specific) magnetic MicroBeads (Miltenyi Biotec, #130–093–634) and microglia were positively selected for via column purification (Miltentyi Biotec, #130-042-201).

Extracted molecule

total RNA

Extraction protocol

Isolated microglia were centrifuged for 5 min at 600xg and resuspended in RLT plus buffer (Qiagen) for extraction and purification of total RNA.
RNA input was normalized and NGS libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

SAL03

Data processing

Sequenced reads were concatenated into files by lane
Concatenated reads were then trimmed using Trimgalore (v.0.6.7) and cutadapt (v.1.18) for low-quality reads, Illumina sequence adaptors were removed, the leading and tailing low-quality base-pairs were trimmed following default parameters
Pair-end reads were mapped to the mm10 genome using STAR (v.2.7.10a) with the following parameters: –outSAMtype BAM SortedByCoordinate –outSAMunmapped Within –outFilterType BySJout –outSAMattributes NH HI AS NM MD XS –outFilterMultimapNmax 20 –outFilterMismatchNoverLmax 0.3 --quantMode TranscriptomeSAM GeneCounts
Resulting bam files were then passed to StringTie (v.2.1.5) to assemble sequenced alignments into estimated transcript and gene count abundance given the NCBI RefSeq GRCm38 (mm10) transcriptome assembly.
Gene count matrices from StringTie were then passed to the R/Bioconductor DESeq2 package (v.1.38.3) for normalization and differential gene expression analyses
Assembly: mm10
Supplementary files format and content: comma separated values file includes gene count matrix output from StringTie with no normalization
Supplementary files format and content: tab-delimited text file contains normalized counts conducted by DESeq2 for differential gene expression analysis

Submission date

Oct 20, 2023

Last update date

Jun 11, 2024

Contact name

Alexander Ve Margetts

E-mail(s)

avm27@miami.edu

Phone

7543666084

Organization name

University of Miami