characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N13-B001633-1_S61

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


status: PUBLIC

tag: attribute

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N13-B001634-1_S145

tag: Namespace:Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: Submitter Id

unit: day

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N13-B001635-1_S229

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N13-B001636-1_S13

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N14-B001633-1_S62

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N14-B001634-1_S146

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N14-B001635-1_S230

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N14-B001636-1_S14

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N15-B001633-1_S63

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N15-B001634-1_S147

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N15-B001635-1_S231

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N15-B001636-1_S15

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N16-B001633-1_S64

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023276:N16-B001634-1_S148

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N16-B001635-1_S232

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N16-B001636-1_S16

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N17-B001633-1_S65

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N17-B001634-1_S149

text: ArrayExpress

text: ependymal cell

text: house mouse

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N17-B001635-1_S233

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N17-B001636-1_S17

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N18-B001633-1_S66

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N18-B001634-1_S150

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N18-B001635-1_S234

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N18-B001636-1_S18

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N19-B001633-1_S67

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023276:N19-B001634-1_S151

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N19-B001635-1_S235

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N19-B001636-1_S19

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N2-B001634-1_S134

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N2-B001635-1_S218

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N20-B001633-1_S68

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N20-B001634-1_S152

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N20-B001635-1_S236

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N20-B001636-1_S20

text: ArrayExpress

text: ependymal cell

text: house mouse

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N21-B001633-1_S69

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023276:N21-B001634-1_S153

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N21-B001635-1_S237

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N21-B001636-1_S21

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N22-B001633-1_S70

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N22-B001634-1_S154

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N22-B001635-1_S238

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N22-B001636-1_S22

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N23-B001633-1_S71

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N23-B001634-1_S155

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N23-B001635-1_S239

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N23-B001636-1_S23

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N24-B001633-1_S65

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023276:N24-B001634-1_S66

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N24-B001635-1_S67

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N24-B001636-1_S68

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N3-B001634-1_S135

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N3-B001635-1_S219

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N4-B001634-1_S136

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N4-B001635-1_S220

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N5-B001634-1_S137

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N5-B001635-1_S221

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N6-B001634-1_S138

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N6-B001635-1_S222

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N7-B001634-1_S139

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N7-B001635-1_S223

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N8-B001634-1_S140

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023276:N8-B001635-1_S224

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023276:N9-B001634-1_S141

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023276:N9-B001635-1_S225

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O1-B001634-1_S157

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O1-B001635-1_S241

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

text: public

text: E-MTAB-115631647023411:O10-B001633-1_S82

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O10-B001634-1_S166

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O10-B001635-1_S250

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O10-B001636-1_S34

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O11-B001633-1_S83

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O11-B001634-1_S167

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O11-B001635-1_S251

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O11-B001636-1_S35

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O12-B001633-1_S84

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023411:O12-B001634-1_S168

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O12-B001635-1_S252

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O12-B001636-1_S36

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O13-B001633-1_S85

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O13-B001634-1_S169

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O13-B001635-1_S253

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O13-B001636-1_S37

text: ArrayExpress

text: ependymal cell

text: house mouse

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O14-B001633-1_S86

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O14-B001634-1_S170

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O14-B001635-1_S254

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O14-B001636-1_S38

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O15-B001633-1_S87

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023411:O15-B001634-1_S171

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O15-B001635-1_S255

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O15-B001636-1_S39

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O16-B001633-1_S88

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O16-B001634-1_S172

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O16-B001635-1_S256

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O16-B001636-1_S40

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O17-B001633-1_S89

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O17-B001634-1_S173

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O17-B001635-1_S257

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O17-B001636-1_S41

text: ArrayExpress

text: ependymal cell

text: house mouse

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O18-B001633-1_S90

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023411:O18-B001634-1_S174

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O18-B001635-1_S258

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O18-B001636-1_S42

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O19-B001633-1_S91

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O19-B001634-1_S175

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O19-B001635-1_S259

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O19-B001636-1_S43

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O2-B001634-1_S158

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O2-B001635-1_S242

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O20-B001633-1_S92

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023411:O20-B001634-1_S176

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O20-B001635-1_S260

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O20-B001636-1_S44

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O21-B001633-1_S93

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O21-B001634-1_S177

text: ArrayExpress

text: ependymal cell

text: house mouse

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O21-B001635-1_S261

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O21-B001636-1_S45

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O22-B001633-1_S94

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O22-B001634-1_S178

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O22-B001635-1_S262

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O22-B001636-1_S46

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O23-B001633-1_S95

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: public

text: E-MTAB-115631647023411:O23-B001634-1_S179

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O23-B001635-1_S263

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O23-B001636-1_S47

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O24-B001633-1_S49

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: not OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O24-B001633-1_S69

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

text: E-MTAB-115631647023411:O24-B001634-1_S70

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK


text: public

text: E-MTAB-115631647023411:O24-B001635-1_S71

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title


characteristic: ENA-CHECKLIST

characteristic: ENA-FIRST-PUBLIC

characteristic: ENA-LAST-UPDATE

characteristic: External Id

characteristic: INSDC center name

text: public

text: E-MTAB-115631647023411:O24-B001636-1_S72

text: ArrayExpress

text: ependymal cell

text: house mouse

text: adult

text: GFP-positive

characteristic: immunophenotype

text: Mus musculus

characteristic: organism

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: OK

characteristic: single cell well quality


characteristic: INSDC first public

characteristic: INSDC last update

text: public

characteristic: INSDC status

characteristic: SRA accession

characteristic: Submitter Id

characteristic: age

text: ArrayExpress

characteristic: broker name

text: ependymal cell

text: house mouse

characteristic: common name

text: Protocols: The ependymal cell layer from the thoracic spinal cord of FOXJ1-EGFP mice was microdissected and dissociated it into a cell suspension. GFP-positive DAPI-negative cells were then sorted into 384-well plates containing lysis buffer. GFP-positive DAPI-negative cells were sorted into lysis plates with a BD Influx Cell Sorter using 6 psi pressure and a 130 μm nozzle. Lysis plates were created by dispensing 0.4 μl lysis buffer (0.5 U Recombinant RNase Inhibitor (Takara Bio, 2313B), 0.0625% Triton X-100 (Sigma, 93443-100ML), 3.125 mM dNTP mix (Thermo Fisher, R0193), 3.125 μM Oligo-dT 30 VN (IDT, 5'AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3') and 1:600,000 ERCC RNA spike-in mix (Thermo Fisher, 4456740)) into 384-well hard-shell PCR plates (Biorad HSP3901) using a Tempest liquid handler (Formulatrix). cDNA synthesis was performed using the Smart-seq2 protocol (Picelli et al., 2013, 2014). Briefly, 384-well plates containing single-cell lysates were thawed on ice followed by first strand synthesis. 0.6 μl of reaction mix (16.7 U/μl SMARTScribe TM Reverse Transcriptase (Takara Bio, 639538), 1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B), 1.67X First-Strand Buffer (Takara Bio, 639538), 1.67 μM TSO (Exiqon, 5'-AAGCAGTGGTATCAACGCAGACTACATrGrG+G-3'), 8.33 mM DTT (Bioworld, 40420001-1), 1.67 M Betaine (Sigma, B0300-5VL), and 10 mM MgCl 2 (Sigma, M1028-10X1ML)) was added to each well using a Tempest liquid handler. Bulk wells received twice the amount of RT mix (1.2 μl). Reverse transcription was carried out by incubating wells on a ProFlex 2x384 thermal-cycler (Thermo Fisher) at 42°C for 90 min and stopped by heating at 70°C for 5 min. Subsequently, 1.6 μl of PCR mix (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3'), and 0.038U/μl Lambda Exonuclease (NEB, M0262L)) was added to each well with a Tempest liquid handler (Formulatrix). The amplified product was diluted with a ratio of 1 part cDNA to 10 parts 10mM Tris-HCl (Thermo Fisher, 15568025), and concentrations were measured with dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). These wells were reformatted to a new 384-well plate at a concentration of 0.3 ng/μl and final volume of 0.4 μl using an Echo 550 acoustic liquid dispenser (Labcyte). If the cell concentration was below 0.3 ng/μl, 0.4 μl of sample was transferred. Illumina sequencing libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131-1096) (Darmanis et al., 2015; Schaum et al., 2018). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then tagmented at 55°C for 10 min. The reaction was stopped by adding 0.4 μl \Neutralize Tagment Buffer\ (Illumina) and spinning at room temperature (RT) in a centrifuge at 3220 xg for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2x384 thermal cycler using the following program: 1. 72°C for 3 minutes, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.

text: adult

text: GFP-positive

text: Mus musculus

text: Mus musculus

text: spinal cord

text: Mus musculus

text: female

text: not OK


text: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee

text: public

text: E-MTAB-115631647023411:O3-B001634-1_S159

text: ArrayExpress

text: ependymal cell

text: house mouse

characteristic: description

text: adult

characteristic: developmental stage

characteristic: genotype

text: GFP-positive

characteristic: individual

text: Mus musculus

text: Mus musculus

text: spinal cord

characteristic: organism part

text: Mus musculus

text: female

characteristic: sex

text: OK

characteristic: strain

characteristic: title