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| Status |
Public on Sep 19, 2021 |
| Title |
O2_g3_63days_S4 |
| Sample type |
SRA |
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| Source name |
O2_g3_63days_S5
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human cerebral organoids
months in culture: 2
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| Growth protocol |
To generate the human cerebral-like organoids we used three hIPSC6-derived lines, one control line (guide against LacZ) and two ZNF558 KD lines (g2 and g3). We used a protocol based by the one by Lancaster et al. with minor modifications (Lancaster et al., 2013). Starting from the plated cells, they were first washed once with DPBS-/-, then detached from the wells with 500 μl of Accutase with an incubation at 37° C for 5 minutes. The cells were then transferred in 5 ml of washing medium (5% KSR in DMEM F12 +/+) and centrifuged for 5 minutes at 400 x g (RT; acceleration/deceleration 8). After removing the supernatant, the pellet was resuspended in 1-2 ml of mTeSR1 (Stemcell Technologies) enriched with ROCK Inhibitor (RI) 10μM (1:1000 from stock concentration 10 mM). The cells in the cell suspension were then counted, and 8000 cells/well were plated in a 96-wells plate (Costar, Ultra Low Attachment, round bottom; REF 7007) with 250 μl of mTeSR1 and RI 10 μM. This is considered day -5 of the differentiation of the iPSCs-derived hFB organoids. At day -3 and -1 the medium was changed (150 μl and 200 μl of mTeSR1, respectively). At day 0 the cells are fed with Neural Induction Medium (NIM; DMEM/F12 media, N2 Supplement (1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:100) and Heparin (2ug/ml).) enriched with 3% KSR. At day 2, 4, and 6, the organoids were fed with NIM with no added KSR. At day 7 “cavities” for the organoids embedding were prepared by putting parafilm on an empty tip holder (with sterile surface facing up) and pressing every hole to form a little basin. The structure was sterilized with a 30-minutes UV cycle and left inside the hood until the next day. Matrigel was left to thaw overnight at 4° C. On day 8 the organoids were embedded: the parafilm was cut in half, then each organoid was transferred in one “cavity” with a cut P1000-tip; the media surrounding the organoid was removed, trying to place it in the center of the “cavity”. The organoids were then covered with 30-50 μl of Matrigel (Corning) avoiding any bubble and incubated at 37° C for 25 minutes to allow the Matrigel to solidify. The organoids were then transferred in Corning REF 3471 6-wells plates with flat bottom containing 3 ml/well of Cortical Differentiation Medium (CDM; F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (-Vit.A, 1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM) and Insulin (2.5 ug/mL)). To detach the organoids from the parafilm, the latter was removed from the tip holder and the “dimples” were turned inside-out; drops of CDM then were used to let the organoids fall into the wells. In the end, in every well there were about 4 ml of medium. On day 10 and 12 of the differentiation, the medium was changed exchanging 3 ml/well for 3 ml of fresh CDM. On day 15, 17, 19, 21 and 23, ~4 ml the medium was replaced with 4 ml of Improved Differentiation Medium + A (IDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A, 1:50), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM)). From day 25, the medium was changed every 3 days with 3-4 ml of Cortical Terminal Differentiation Medium (CTDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A) – (1:50) 800uL, L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM), BDNF (10ng/uL), cAMP (200uM), GDNF (10ng/uL)). All the diameter measurements of the organoids were taken with the Measure tool from the software GIMP 2.10. The chosen measuring unit was mm.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Organoids (LacZ, g2 and g3) were collected for single nuclei sequencing at 2 and 4 months. At harvest, organoids were dissected out of the Matrigel, frozen on dry ice and stored at -80°C. In order to reduce bias in the downstream analysis due to organoid heterogeneity, they were frozen 5 by 5 per time point and batch. The nuclei isolation protocol has been described in detail elsewhere (Sodersten et al., 2018). In brief, the organoids were thawed and homogenized in cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose solution (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before resuspended in 400 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C with low flowrate using a 100 mm nozzle (reanalysis showed >99% purity), sorting 8,500 nuclei per sample which were then loaded onto 10X Genomics Single Cell 3’ Chip along with the reverse transcription mastermix following the manufacturer’s protocol for the Chromium Single Cell 3′ Library (10X Genomics, PN-120233) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics and sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples were multiplexed and sequenced on a Novaseq using a 150-cycle kit using the recommended read length.
Chromium Single Cell 3′ (10x)
single nuclei RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Organoids (LacZ, g2 and g3) were collected for single nuclei sequencing at 2 and 4 months. At harvest, organoids were dissected out of the Matrigel, frozen on dry ice and stored at -80°C. In order to reduce bias in the downstream analysis due to organoid heterogeneity, they were frozen 5 by 5 per time point and batch. The nuclei isolation protocol has been described in detail elsewhere (Sodersten et al., 2018). In brief, the organoids were thawed and homogenized in cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose solution (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before resuspended in 400 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C with low flowrate using a 100 mm nozzle (reanalysis showed >99% purity), sorting 8,500 nuclei per sample which were then loaded onto 10X Genomics Single Cell 3’ Chip along with the reverse transcription mastermix following the manufacturer’s protocol for the Chromium Single Cell 3′ Library (10X Genomics, PN-120233) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics and sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples were multiplexed and sequenced on a Novaseq using a 150-cycle kit using the recommended read length.
human cerebral organoids
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| Data processing |
single-nuclei RNA-seq: raw basecall files were converted to fastq and demultiplexed using cellranger/2.1 mkfastq. Gene counting was done with cellranger count.
Genome_build: hg38
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| Submission date |
Sep 14, 2021 |
| Last update date |
Sep 19, 2021 |
| Contact name |
Johan Jakobsson |
| E-mail(s) |
johan.jakobsson@med.lu.se
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| Organization name |
Lund University
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| Department |
Wallenberg Neuroscience Center
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| Lab |
Molecular Neurogenetics
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| Street address |
Sölvegatan 17
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| City |
Lund |
| ZIP/Postal code |
22362 |
| Country |
Sweden |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE182224 |
Structural variation at the ZNF558 locus controls a gene regulatory network in human brain development |
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| Relations |
| BioSample |
SAMN21435968 |
| SRA |
SRX12188867 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5578198_O2_g3_63days_Hg38_barcodes.tsv.gz |
12.1 Kb |
(ftp)(http) |
TSV |
| GSM5578198_O2_g3_63days_Hg38_features.tsv.gz |
297.6 Kb |
(ftp)(http) |
TSV |
| GSM5578198_O2_g3_63days_Hg38_matrix.mtx.gz |
11.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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