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Sample GSM8144273

Query DataSets for GSM8144273

Status

Public on May 20, 2024

Title

6Bone Marrow-BCG

Sample type

SRA

Source name

Bone Marrow

Organism

Mus musculus

Characteristics

tissue: Bone Marrow
cell line: Bone Marrow cells
cell type: Bone Marrow cells (unsorted)
genotype: wt
treatment: BCG

Growth protocol

Mice were subcutaneously vaccinated with 10^6 CFU of Mycobacterium bovis/BCG in 100 µl phosphate-buffered saline, mice in the control group were subcutaneously injected with 100 µl of PBS. Three days after BCG administration, vaccinated and control animals were sacrificed under isoflurane anesthesia, by exsanguination and subsequent cervical dislocation, after which spleen and bone marrow were collected. Single-cell suspensions from the organs were stained with conjugated anti-CD45, anti-CD11b, anti-NK1.1, anti-Ly6G, and anti-Ly6C antibodies, and subjected the suspensions to fluorescence-activated cell sorting (FACS). The prepared cell suspensions were used for FACS sorting and three cell populations were collected: monocytes [CD45+CD11b+Ly6C+], neutrophils [CD45+CD11b+Ly6G+] and NK-cells [CD45+CD11b-NK1.1+]. Moreover, 1 million of total unsorted bone marrow cells from each mouse were collected. The samples were collected for each mouse from both control and BCG vaccinated groups.

Extracted molecule

polyA RNA

Extraction protocol

Harvested cells were lysed using ExtractRNA (Evrogen, Moscow, Russian Federation). RNA was extracted with RNA Clean & Concentrator TM-5 (Zymo Research, Irvine, CA, USA). To get rid of the excess ribosomal RNA, NEBNext rRNA Depletion Kit v2 was used.
The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with Sample Puri-fication Beads (New England BioLabs) and a NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1, New England BioLabs) were used for the preparation of libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

The raw reads were mapped on the murine reference genome (mm10) using HISAT2 (Galaxy Version 2.2.1+galaxy1)
The featureCounts tool (Galaxy Version 2.0.3+galaxy2) was used for counting reads to GENCODE release M23 annotated genes.
Assembly: mm10
Supplementary files format and content: .txt format, matrix table with featureCounts read counts

Submission date

Mar 12, 2024

Last update date

May 20, 2024

Contact name

Liya Kondratyeva

E-mail(s)

liakondratyeva@yandex.ru

Organization name

Institute of bioorganic chemistry of the Russian Academy of Sciences

Street address

Ulitsa Miklukho Maklaya, 16/10