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Sample GSM7794405

Query DataSets for GSM7794405

Status

Public on Jun 27, 2024

Title

KO_7.6_DMSO_6h_2

Sample type

SRA

Source name

Bone Marrow Derived Denderitic Cell

Organism

Mus musculus

Characteristics

tissue: Bone Marrow Derived Denderitic Cell
genotype: GPR65 KO Homozygous
age: 6.5 weeks
treatment: KO
treatment: Stimulated for 6 hours at pH 7.6 using DMSO

Treatment protocol

For stimulation, media was removed and replaced with 80uL DMEM (10% heat-inactivated FBS, GlutaMAX, 1% penicillin-streptomycin), no rmGM-CSF, adjusted to pH 6.6, 7.2, or 7.6. Each well then received 10uL of media w/ zymosan and 10uL of DMSO/PGE2/BRD5075. Final concentrations: 5uM PGE2, 20uM BRD5075, 5ug/mL Zymosan, 0.25% DMSO. Stimulated for either 2 or 6 hours at 37C, 5% CO2.

Growth protocol

BMDCs were differentiated from bone marrow in DMEM (10% heat-inactivated FBS, GlutaMAX, 1% penicillin-streptomycin) containing 20ng/mL rmGM-CSF (Peprotech) for 7 days, supplemented with additional DMEM + rmGM-CSF on Day 3. Cells were then scraped and seeded at 1e5 cells per well in a 96-well microplate and allowed to adhere overnight.

Extracted molecule

total RNA

Extraction protocol

Centrifuge plates at 100xg for 3 mins, remove supernatant. Wash 1X with 100uL PBS, aspirate. Add 100uL lysis buffer, pipet up and down and freeze at -80C.
Libraries were constructed using a modified SmartSeq2 protocol.RNA lysate were purified with Dynabeads mRNA Direct Purification kit (Life Technologies),followed by reverse transcription with Maxima Reverse Transcriptase (Life Technologies) and whole transcription amplification (WTA) with KAPA HotStart HiFi 2x ReadyMix (Kapa Biosystems).WTA products were purified with Ampure XP beads (Beckman Coulter). RNA-Seq libraries were constructed using Nextera XT DNA Library Preparation kit (Illumina).
Libraries were sequenced with Nextseq500(75cycles) paired-end and the following parameters : paired End: Index1 8, Index2 8, Read1 38, Read2 38

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Alignment with STAR
Followed by RSEM
Downstream analysis with custom R and Python script
Assembly: mm10
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample in csv file format

Submission date

Sep 20, 2023

Last update date

Jun 27, 2024

Contact name

Ramnik Xavier

E-mail(s)

xaviergeo@broadinstitute.org

Organization name

Broad Institute of MIT and Harvard

Lab

Xavier Lab