organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA13364721
attributes.tag: INSDC center alias
attributes.value: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee
attributes.tag: INSDC center name
attributes.value: Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:59Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11563:D15-B001634-1_S207
attributes.tag: age
attributes.value: 85
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: ependymal cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: Tg(FOXJ1-EGFP)85Leo
attributes.tag: immunophenotype
attributes.value: GFP-positive
attributes.tag: individual
attributes.value: 335865
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: spinal cord
attributes.tag: sample name
attributes.value: E-MTAB-11563:D15-B001634-1_S207
attributes.tag: sex
attributes.value: female
attributes.tag: single cell well quality
attributes.value: OK
attributes.tag: strain
attributes.value: B6; C3-Tg(FOXJ1-EGFP)85Leo/J