Myelomeningocele spinal cord organoids scRNAseq

Myelomeningocele (MMC) is a severe form of spina bifida associated with substantial neurologic morbidity. In vitro modeling systems of human spinal cord development may help to elucidate the underlying pathophysiology of the MMC spinal cord. We developed spinal cord organoids (SCO), defined as self-organized, three-dimensional clusters of spinal tissue, that were derived from human amniotic fluid induced pluripotent stem cells. Here, we used a variety of analyses, including immunofluorescent and single-cell transcriptomic approaches, to compare SCOs from healthy and MMC fetuses. Organoids contained a diverse range of neural and mesodermal phenotypes when cultured for up to 130 days in vitro. Multielectrode arrays revealed functional activity with evidence of emerging neuronal networks. Fetal spina bifida modeling was successfully established by culturing MMC SCOs in second- and third-trimester amniotic fluid for 3 weeks. Taken together, we show that functional SCOs can recapitulate the ..., Single-cell RNA sequencing library preparation
scRNA-seq libraries were generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index). Per manufacturer’s protocol, a maximum volume of 43.3 µL/sample was used for processing to target up to 10,000 cells. Cells were combined with RT reagents and loaded onto 10X Next GEM Chip G along with 3’ v3.1 gel beads. Approximately 100µL of emulsion was retrieved from the chip and incubated (45 min at 53°C, 5 min at 85°C, cool to 4°C), generating barcoded cDNA from each cell. Samples were cleaned using 0.6X SPRIselect beads. Then 10uL of amplified cDNA was carried into library preparation. Fragmentation, end repair, and A-tailing were completed and samples were cleaned up with SPRIselect beads. Adaptor ligation was followed by a 0.8X cleanup, and then amplification was performed with PCR. Libraries were sequenced on the Illumina NovaSeq 6000 using v1.5 kits, targeting 50K reads/cell. Demultiplexing and FASTQ generation was comple..., , # **README: SCO scRNAseq**

Data for *Spinal Cord Organoids from Human Amniotic Fluid iPSC Recapitulate the Diversity of Cell Phenotypes During Fetal Neural Tube Morphogenesis*

Single-cell RNA sequencing data of spinal cord organoids generated from amniotic fluid cells collected from one control and two myelomeningocele fetuses.

We have uploaded 3 stages of the data for this paper:

1. Raw FASTQ files generated & demultiplexed with Illumina's BaseSpace software. Organoid libraries prepped with Chromium Next GEM Single Cell 3' Reagent KIits v3.1 (Dual Index) and sequenced with Illumina NovaSeq 6000, targeting 50K reads/cell.
2. UMI Count Matrices from aligning FASTQ files to GRCh38 human genome reference assembly with 10X Genomics Cell Ranger 6.1.1.
3. Processed & clustered SeuratObject with identified cell types & metadata.

## **Description of the data and file structure**

The four cell lines are labeled as follows: H29_5, H42_2, H47_4, and H47_62.

Sample H42_2 is the Control, all ...,