characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-11-02T00:13:04Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-11-02T00:13:04Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA10944896
characteristics: External Id
characteristics: Institut of Humangenetics, University Hospital Jena, Jena, Germany
characteristics: INSDC center name
characteristics: 2022-11-02T00:13:04Z
characteristics: INSDC first public
characteristics: 2022-11-02T00:13:04Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS8594807
characteristics: SRA accession
characteristics: E-MTAB-11147:C-KO-neg1
characteristics: Submitter Id
characteristics: 23
characteristics: week
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: neural cell
characteristics: cell type
characteristics: house mouse
characteristics: common name
characteristics: Protocols: For the analysis of the transcriptome of PV-positive interneurons, animals at the age of 23 weeks were sacrificed by cervical dislocation and decapitated. The brain was quickly removed with sterile instruments and placed in Grey’s Balanced Salt Solution with glucose (GBSS/Glc: 137.93 mM NaCl, 3.66 mM KCl, 2.702 mM NaHCO3, 1.03 mM CaCl2, 0.28 mM MgSO4x7H2O, 0.84 mM Na2HPO4, 0.22 mM KH2PO4, 5.56 mM glucose; pH 7.4; sterile filtered) on ice. The cortex and the hippocampus were dissected in GBSS/Glc on ice. The tissue was cut into small pieces (1 - 2 mm3) with a sterile razor blade and transferred into falcons in a sterile environment. After washing (gentle shaking; wash medium 98.2 ml HBSS w/o Ca and Mg; 700 µl 1M HEPES; 1 ml Penicillin-Streptomycin; 100 µl 65 % glucose) the tissue was incubated for 5 min with 0.25 % Trypsin/EDTA (Thermo Fisher Scientific; 4.5 ml 0.25 % Trypsin/EDTA, 50 µl Penicillin-Streptomycin, 50 µl 1M HEPES, 500 µl 50% trehalose) for 30 min at 37°C. Pre-incubated DNAse (AplliChem, 50 µl, 4 µg/µl, 600 U) was added to the mix. A washing step with 2.1 ml trypsin stop-medium (40 ml DMEM/F12, 5 ml FBS, 500 µl Penicillin-Streptomycin, 5 ml 50 % trehalose) led to the inactivation of trypsin. 900 µl collagenase type 2 (Worthington, 10 mg/ml in HBSS) was added to the medium and the tissue was incubated again for 25 min at 37°C. After another washing step with warm trypsin stop-medium and 3.63 µl 0.5 M EDTA, the samples were left on ice to cool down and finally resuspended in 1.5 ml trypsin stop-medium. Sterile coated (Sigmacote®) Pasteur pipettes with three different opening diameters (large, medium and small) were used to homogenize the tissue (pipetted up and down max 20 times). The lysate was centrifuged at 4°C (5 min, 160 rcf) and resuspended in 4 ml wash-buffer/trehalose (36 ml HBSS w/o Ca and Mg, 280 µl 1M HEPES, 400 µl Penicillin-Streptomycin, 40 µl 65 % Glucose, 4 ml 50 % trehalose). After another centrifugation step (4°C, 5 min, 160 rcf), the pellet was resuspended in 4 ml 30 % Percoll™ (GE Healthcare, 7.5 ml Percoll™, 15 ml PBS, 2.5 ml 50 % trehalose) for a density gradient centrifugation (4°C, 13 min, 600 rcf). The supernatant and the cell waste were discarded in order to resuspend the pellet in 1.0 ml wash-buffer/trehalose. Pellets were resuspended in 1.0 ml wash-buffer/trehalose and sorted. Cell suspensions prepared from the cortical hemispheres and hippocampi of 23-week-old mice were subjected to FACsorting. Following addition of DAPI, cells were sorted using an ARIA III FACS sorter (BD Biosciences, United States) with a maximal flow rate of six. The tdTomato reporter was excited by a laser (excitation 554 nm, emission 581 nm). Living cells were sorted based on a distinctive tdTomato signal. Two populations were sorted: tdTomato-positive (+) cells and tdTomato-negative (-) cells. The different cell populations (Co-tomato+; Co-tomato-; HC-tomato+; HC-tomato-) were collected in 1xPBS with 2 % FBS and centrifuged (4°C; 10 min; 1000 g). The supernatant was discarded and the cells were lysed in 150 µl TRIzol™ Reagent (Thermo Fisher Scientific) and then frozen (-80 °C) for further processing. RNA was isolated and purified using the Direct-zol™ RNA MicroPrep Kit from Zymo Research. All samples were processed according to the manufacturer’s protocol and checked for integrity by capillary gel electrophoresis (Bioanalyzer, Agilent Technologies, Inc., United States). The RNA samples were stored at -80°C. Total RNA of cells and native controls were used in biological triplicates to perform whole transcriptome analysis. Library preparation for RNA-Seq was performed using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara, Cat# 634412) including rRNA-depletion and further processed according to the manufacturer’s protocol. The concentrations of final cDNA libraries were determined using the Qubit 2.0 fluorometer (Invitrogen) (average 20 ng/µl) and quality checked with a Bioanalyzer (Agilent 2100 Bioanalyzer) high sensitivity DNA assay.
characteristics: description
characteristics: adult
characteristics: developmental stage
characteristics: KCC2 knockout
characteristics: genotype
characteristics: Mus musculus
characteristics: organism
characteristics: Mus musculus
characteristics: organism
characteristics: brain
characteristics: organism part
characteristics: cortex
characteristics: sampling site
characteristics: Mus musculus
characteristics: scientific_name
characteristics: not available
characteristics: sex
characteristics: C57BL/6
characteristics: strain
characteristics: C-KO-neg1
characteristics: title