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Sample GSM5606718

Query DataSets for GSM5606718

Status

Public on Jan 01, 2022

Title

ST3D-C4

Sample type

SRA

Source name

ST3Ds

Organism

Macaca mulatta

Characteristics

tissue: placental
treatment: uninfected control
Sex: female

Treatment protocol

For infection of the TSCs and EVTs, one flask of cells from each line was lifted and counted just prior to infection. For TSCs and EVTs, media were aspirated, and 1 ml of inoculum was added to the flask. Cultures were incubated at 37°C and rocked gently every 15 mins for 1 hr. The inoculum was removed, and the cells were washed once with PBS followed by addition of fresh medium. The number of ST3D “cells” (at this point they are aggregates) present was based on the number of TSCs added to the flask three days prior. To infect the ST3Ds that were grown in suspension, aggregates/cells were pelleted by centrifugation at 500 x g for 3 mins, the supernatant was removed, and the cells were resuspended in 300 µl of inoculum. Aggregates were incubated in the ZIKV inoculum for 2 hrs at 37°C and gently disturbed every 30 mins. Since ST3Ds are grown in suspension, a larger volume of inoculum was used, and the length of inoculation was extended to account for this increase. Next, 2 ml of PBS was added, the cells were re-pelleted by centrifugation as above, and the supernatant was removed. A volume of 10 ml ST3D medium was added to the cells, and they were then transferred back into a T75 flask. Once fresh media were added back for all cell types, a 500 µl aliquot was removed to serve as a baseline of initial viral titer in the culture. The aliquot was spun at 500 x g for 5 mins to remove any cellular debris and a plaque assay was conducted. For EVTs, Growth Factor Reduced Matrigel (0.5%; Corning, Cat #354230) was supplemented to the cultures after this step. EVTs were cultured for a day longer than previously reported 11 (72 hrs total) to extend the duration of ZIKV infection.

Growth protocol

TSCs were differentiated into either EVTs or ST3D aggregates TSCs were grown in 13 ml media, EVTs in 15 ml media, and ST3Ds in 10 ml media. TSCs were differentiated to EVT by seeding into T75 flasks coated with 1 µg/ml collagen (col IV; Corning, NY, USA, Cat #354233) with 2% Matrigel (Corning, Cat # 354234) added to the EVT media. No additional Matrigel was added on day 3 when the media were changed as previously described. Growth Factor Reduced Matrigel (0.5%; Corning, Cat #354230) was added on day 6. For ST3D culture, TSCs were plated in non-adherent T75 flasks (Thermo Fisher, Cat #174952 and Cat #156800) and cultured for 5 days total. Three days after initial plating, the media were removed, and fresh media added. For additional details on the media composition and growth conditions, please see Schmidt, et al. 2020 11.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated from cells using an RNeasy kit (Qiagen, Cat #74104) following kit recommendations with modifications. A volume of 700 µl of Qiazol was added to the cell pellet and then frozen at -80°C until RNA extraction. The protocol was followed with the same minor adaptations as with the miRNeasy kit. A 15 min DNAse treatment was performed on the column using RNase-Free DNase (Qiagen, Cat # 79254) prior to washing the column.
The poly(A) RNA cDNA sequencing library was prepared following Illumina’s TruSeq-stranded-mRNA sample preparation protocol. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Pair-end sequencing reads of 150 bp reads were generated on an Illumina NovaSeq 6000 sequencing system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

For cell poly(A)seq data, sequencing reads were filtered to remove adaptors and primer sequences and to remove sequences with a quality score lower than 20.
The cleaned sequencing reads were aligned to the reference genome (GCF_003339765.1_Mmul _10) using the HISAT2 package. Multiple alignments with a maximum of two mismatches were allowed for each read sequence (up to 20 by default).
Transcript abundance estimation and differential expression analysis of aligned reads of individual samples were assembled using StringTie.
Transcriptomes from all samples were then merged to reconstruct a comprehensive transcriptome using a proprietary Perl script designed by LC Sciences.
Following transcriptome reconstruction, raw read counts were filtered, normalized, and differential expression determined with DESeq2 and edgeR. The glmLRT test was used for edgeR differential expression analysis
Genome_build: GCF_003339765.1_Mmul _10
Supplementary_files_format_and_content: alldata_IPA.csv contains fold change, p-value, and counts per million (CPM) for all the cell types analyzed by gene ID. The my.dge.csv file contains the normalized counts.

Submission date

Sep 30, 2021

Last update date

Apr 07, 2022

Contact name

Thaddeus Golos

E-mail(s)

golos@primate.wisc.edu

Organization name

UW Madison

Department

WNPRC