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Sample GSM5558483

Query DataSets for GSM5558483

Status

Public on Aug 31, 2022

Title

CoVinh_1

Sample type

SRA

Source name

AT2_organoids

Organism

Homo sapiens

Characteristics

cell_origin: AO24
infection: mock
tretament: CTR

Treatment protocol

Organoid fragments were either mock-infected or challenged with SARS-CoV-2 (MOI 1, ) Munich/2020/984 strain). After incubation at 37° and 5 % CO2 for 30 min, medium with the respective inhibitor or carrier (PH-797804, VX-702, or DMSO only) was added.

Growth protocol

For generation of adult stem cell derived lung organoids, primary cells were isolated from healthy parts of distal lung tissue obtained from lung cancer patients undergoing tumor resection surgery, cells were FACS sorted, counted and seeded in the extracellular matrix substitute. Organoids were kept in an incubator at 37°C, 5 % CO2. Upon growth for 2-3 weeks, the organoids were expanded by enzymatic digestion. Mature human lung organoids were collected on ice to remove remaining matrix.

Extracted molecule

total RNA

Extraction protocol

For total RNA isolation, the RNeasy Plus Mini Kit (Qiagen) was used according to the manufacturer`s instructions. Organoids were released from Cultrex with cold PBS, pelleted, and resuspended by vortexing in 350 μl RLT lysis buffer supplemented with β-mercaptoethanol. RNA was eluted in 30 µl water.
PolyA+RNA was purified from 200 ng total RNA using Poly(A) mRNA Magnetic Isolation module Kit (NEB E7490L, New England Biolabs). The RNA Sequencing library was prepared with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs).
The libraries were sequenced on Illumina Nextseq 2000 using NextSeq2000 P3 Reagent Kit (200 cycles, paired end run 2x 111 bp) with an average of 74.2 M reads per RNA sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Data processing

Reads were quality checked with package FastQC (version 0.11.4,), then trimmed using Trimgalore (version 0.4.4) with default settings.
Trimmed reads were mapped to human genome ENSEMBL hg38 release 91 and to CoV-2 genome EPI_ISL_463008_Cov2_AF, using STAR aligner (Dobin and Gingeras, 2015) with default settings.
Mapped reads were counted using RsubRead (version 1.32.4, (Liao et al., 2019)). Raw counts were then normalized using DESeq2 (version 1.16.1, (Love et al., 2014)).
Genome_build: ENSMBL human hg38 release 91 genome and CoV-2 genome EPI_ISL_463008_Cov2_AF
Supplementary_files_format_and_content: txt matrix with log2 normalized values per sample.

Submission date

Sep 06, 2021

Last update date

Aug 31, 2022

Contact name

Klaus Schughart

E-mail(s)

labschughart@online.de

Phone

+49-159-0483-7911

Organization name

University Münster