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Sample GSM8409415

Query DataSets for GSM8409415

Status

Public on Aug 31, 2024

Title

OrgNK_S_6.2_6.1

Sample type

SRA

Source name

placenta_decidua

Organism

Homo sapiens

Characteristics

tissue: placenta_decidua
cell line: OrgNK_S_6.2_6.1
cell type: trophoblast_dNK
genotype: NA
treatment: sorted
batch: 1

Treatment protocol

When co-cultures with dNKs or dNK monocultures were prepared, 50 000 dNKs were added to each 40mL Cultrex dome. This number of dNKs was selected because it was the largest number that could be accommodated before their presence caused the Cultrex gel to depolymerize.

Growth protocol

Trophoblast organoids were cultured according to the protocol described by (Turco et al., 2018), with the following modifications: mouse HGF (Sino Biological 50038-MNAH) rather than human HGF was used, cultures were performed using reduced growth factor Cultrex basement membrane extract (R&D #3433), and cultures were maintained in hypoxia chambers (Billups-Rothenberg MIC-101) perfused with 2% O2, 5% CO2 for 5 minutes at a rate of 25 L/min. Media was pre-conditioned in the perfused hypoxia chamber in a 37oC incubator for one hour before being applied to cultures. Trophoblast organoid media was comprised of: 1X N2 supplement (Gibco 17502048), 1X B27 supplement minus vitamin A (Gibco 12587010), 2mM L-glutamine (R&D B90010), 1.25 mM N-Acetyl-L-Cysteine (Cayman Chemical 20261), 50ng/mL human EGF (Gibco PHG0311L), 100ng/mL human FGF2 (Sigma-Aldrich F0291), 50ng/mL mouse HGF (Sino Biological 50038-MNAH), 80ng/mL human R-spondin (R&D 4645-RS), 500nM A83-01 (Tocris 2939), 2.5uM prostaglandin E2 (Tocris 2296), 1.5uM CHIR99021 (Cell Signaling Technology #54290S), 2uM Y27632 (Cell Signaling Technology #13624), and 1X Antibiotic-Antimycotic (Gibco 15240062) added to Advanced DMEM/F12 (Gibco 12634010). Trophoblast organoid cultures were initiated by seeding each dome of Cultrex gel either with <50 000 of thawed primary trophoblast cells or with thawed cryopreserved organoids from previous cultures. 40mL domes of Cultrex gel containing cultures were prepared in 24-well plates, allowed to polymerize upside-down, and were then overlaid with 500mL of trophoblast organoid media per dome.

Extracted molecule

polyA RNA

Extraction protocol

All samples were collected and snap frozen following seven days of culture, then processed according to the manufacturer’s instructions using the RNeasy Micro Kit (Qiagen 74004) for total RNA isolation. The sample disruption and homogenization steps were performed using QIAShredders (Qiagen 79654).
Libraries were prepared at the Donnelley Centre at the University of Toronto using Takara SMART-Seq v.4 Ultra Low Input RNA kits.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq X

Data processing

FASTQ files were quality assessed by FASTQC, filtered for low quality reads and adapter sequences using cut adapt. Cleaned reads were aligned using HISAT2 (2.2.1), against the human genome build GRCH37. Sam files were converted to BAM files, sorted and indexed using samtools (1.13, htlib 1.13+ds). R (4.1) scripts were used to create a count table from BAM files and the human transcriptome G (including coding and non-coding RNA species) using the featureCounts function of Rsubread (2.8.2). Differential expression was calculated using a pipeline of EdgeR (4.2.1), and Limma (3.60.3) packages. Gene set enrichment was calculated by the camera function in the limma package. Human gene ontology files were obtained from the enrichment map resource (download.baderlab.org/EM_Genesets/). Human placental ontologies were from supplemental files from Naismith and Cox, 2021. Barcode graphs were generated using the barcode function in limma. Removal of dNK gene expression was accomplished using the granulator package in R applying the detanglr algorithm to estimate the transcriptional contribution of the NK cells to the co-cultured bulk mRNA signal. The dNK and organoid samples were used to estimate the pure signal. The estimated mean contribution was removed in limma as a fitted model.
Assembly: GRCH37
Supplementary files format and content: table of raw counts

Submission date

Jul 19, 2024

Last update date

Aug 31, 2024

Contact name

Brian Joseph Cox

E-mail(s)

b.cox@utoronto.ca

Organization name

University of Toronto

Department

Physiology