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Sample GSM4635988

Query DataSets for GSM4635988

Status

Public on Dec 07, 2020

Title

SmallAirway_1_SARS-CoV-2

Sample type

SRA

Source name

human small airway 2D culture infected with SARS-CoV-2 for 72 hours

Organism

Homo sapiens

Characteristics

cell type: Small airway
treatment: SARS-CoV-2
genotype: Wildtype

Treatment protocol

2D differentiated cultures were washed twice with 200 ul AdDF+++ before inoculation from the apical side at a MOI of 0.01 or 0.1 (as indicated) in 200 μL AdDF+++ per well. Next, cultures were incubated at 37°C 5% CO2 for 2 hours before washing 3 times in 200 μL AdDF+++.

Growth protocol

Airway organoids were passaged by mechanical disruption at 1:2 to 1:4 ratios after 10–14 days of culture. Airway organoid medium (Sachs et al., 2019) was changed every 4 days. To obtain differentiated organoid-derived cultures, organoids were dissociated into single cells using TrypLE express (Gibco; #12604013). Cells were seeded on Transwell membranes (Corning) coated with rat tail collagen type I (Fisher Scientific). Single cells were seeded in AO growth medium : complete base medium (CBM; Stemcell Pneumacult-ALI; #05001) at a 1:1 ratio. After 2-4 days, confluent monolayers were cultured at air‐liquid interphase in CBM. Medium was changed every 5 days. After 8 weeks of differentiation, cultures were used for infection experiments. Fetal lung bud tip organoids were passaged by mechanical disruption at 1:2 to 1:4 ratios after 10–14 days of culture. Fetal lung (FL) medium (Nikolic et al., 2017) was changed every 4 days. To obtain differentiated organoid-derived cultures, 105 donor-specific fetal lung mesenchymal cells were seeded in 12-well plates in AdDF+++ containing 10% FCS. The next day, organoids were dissociated into small clumps using TrypLE express. Cells were seeded on Transwell membranes (Corning‐3460). Clumps were seeded in FL : BEpiCM (bronchial epithelial cell growth medium; Sciencell; Cat. No. 3211) at a 1:1 ratio. BEpiCM was made by mixing basal medium (Sciencell; Cat. No. 3211-b) and DMEM at a 1:1 ratio and adding bronchial epithelial cell growth supplement (BEpiCGS, Sciencell; Cat. No. 3262), penicillin (100 IU/mL), streptomycin (100 IU/mL) and 0.001 μM retinoic acid. ROCK inhibitor was added for the first 5 days to prevent apoptosis (Y-27632; Sigma; Y0503; 10µM). After 4-7 days, confluent monolayers were cultured at air‐liquid interphase in BEpiCM. Eighty percent of the medium was changed every 4-5 days. After 14 days of differentiation, cultures were used for infection experiments.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the standard TRIzol (Invitrogen) protocol and used for library preparation and sequencing.
mRNA was processed as described previously, following an adapted version of the single-cell mRNA seq protocol of CEL-Seq (Hashimshony et al., 2016; Simmini et al., 2014). In brief, samples were barcoded with CEL-seq primers during a reverse transcription and pooled after second strand synthesis. The resulting cDNA was amplified with an overnight In vitro transcription reaction. From this amplified RNA, sequencing libraries were prepared with Illumina Truseq small RNA primers.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

demultiplexed.38.CS2C8U6
Bulk RNA sequencing of human small airway 2D culture infected with SARS-CoV-2 for 72 hours

Data processing

Paired end reads were aligned to the transcriptome using bwa. Paired end reads obtained by CEL-seq were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all RefSeq gene models based on the human genome release hg38. The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first eight bases correspond to the cell specific barcode followed by 4 bases representing the unique molecular identifier. The remainder of the left read contains a polyT stretch followed by few (<15 transcript derived bases). The left read was not used for quantification. For each cell barcode we counted the number of unique molecular identifiers for every transcript and aggregated this number across all transcripts derived from the same gene locus.
Genome_build: hg38
Supplementary_files_format_and_content: raw counts; UMI deduplicated, annotation pooled, normalized abundance counts

Submission date

Jun 25, 2020

Last update date

Dec 07, 2020

Contact name

Jelte van der Vaart

E-mail(s)

j.vaart@hubrecht.eu

Organization name

Hubrecht Institute

Lab

dr. Hans Clevers Lab