characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112647605
characteristics.characteristic: External Id
characteristics.text: Department of Hematology Erasmus MC
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14645334
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12615:BL6_exp10_nr9_d37_CD8
characteristics.characteristic: Submitter Id
characteristics.text: 12
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: cytotoxic T cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: For bone marrow stromal cells two femurs and two tibias from each mouse were used. The ends of cleaned bones were cut off and marrow was flushed out. The remaining bone was cut into small pieces and gently crushed with a mortar and pestle. Bone fragments were filtered through a 100 um cell strainer and then digested for 45 minutes at 37C with 0.25% Collagenase Type I (Stem Cell). Bone chips were again washed on a 100 um cell strainer and stromal cells were collected into PBS containing 1% FCS and 1 mM EDTA. Erythrocytes were lysed and cells were counted. For bone marrow immune cells 1 femur and 2 tibias from each mouse were used. After isolating and cleaning the bones, one end was cut off and bones were placed into a 0.5 ml microcentrifuge tube with a hole punched in the bottom. This was nested into a 1.5 ml microcentrifuge tube and spun down at 10,000 RPM for 15 seconds to collect the bone marrow. Following resuspension in PBS containing 1% FCS and 1 mM EDTA, erythrocytes were lysed. Remaining cells were washed with PBS containing 2% FCS, filtered through a 100 um cell strainer and counted. For all flow cytometry and FACS experiments, Fc-receptors were blocked with 10% normal mouse serum and anti-CD16/32 (BD, clone 2.4G2) prior to incubation with the following antibodies: CD3e-PerCpCy5.5 (eBioscience, clone 145-2C11), CD8-FITC (BD, clone 53-6.7), CD11b-APCeFluor780 (eBioscience, clone M1/70), CD19-AF700 (eBioscience, clone eBio1D3), CD27-BV510 (Biolegend, clone LG.3A10), CD31-PE/CF594 (BD, clone MEC13.3), CD44-BV510 (Biolegend, clone IM7), CD45-APC/Cy7 (eBioscience, clone 30-F11), CD51-PE (eBioscience, clone RMY-7), CD69-AF647 (Biolegend, clone H1.2F3), Biotinylated CD71 (Biolegend, clone R17217), NKp46-PE-Cy7 (eBioscience, clone 29A1.4), Gr1-APC (BD, clone RB6-8C5), Ly6c-BV510 (Biolegend, clone HK1.4), Sca-1-PerCP/Cy5.5 (Invitrogen, clone D7), Ter119-BV510 (Biolegend, clone TER-119), Streptavidin-PE/Cy7 (Biolegend). Samples were incubated for 25 minutes on ice in the dark. Following staining, cells were washed and resuspended in PBS + 2% FCS containing DAPI, and sorted with a BD FACS Aria III. RNA was extracted using the NucleoSpin RNA XS kit (Machery‐Nagel). cDNA was prepared using SMARTer Ultra Low RNA kit (Clontech Laboratories) for Illumina Sequencing according the manufacturer’s protocol. The quality and quantity of cDNA samples was verified using the Agilent 2100 Bio-Analyzer and the High Sensitivity DNA kit. cDNA Libraries were generated using the TruSeq Sample Preparation v.2 Guide (Illumina).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: multiple myeloma
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: Exp10_mm_B_9
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: bone marrow cell
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: C57Bl/6JOlaHsd
characteristics.characteristic: strain
characteristics.text: BL6_exp10_nr9_d37_CD8
characteristics.characteristic: title