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Sample GSM6939361

Query DataSets for GSM6939361

Status

Public on Sep 20, 2023

Title

WA_WR_0726_4

Sample type

SRA

Source name

ADGRG6 gRNA +dox Air_biol rep 1

Organism

Homo sapiens

Characteristics

tissue: Cell line
cell line: BU3 NGST CRISPRi line
cell type: iPSC-derived AT2
genotype: ADGRG6-kd
treatment: CK+DCI media +doxycyline; air-exposed

Growth protocol

To generate iAT2s, we performed PSC directed differentiation via definitive endoderm into NKX2-1 lung progenitors using methods we have previously described (Hawkins et al., JCI 2017; Jacob et al., Cell Stem Cell 2017; Jacob et al., Nat Protoc 2019). In brief, cells maintained in mTeSR1 media were differentiated into definitive endoderm using the STEMdiff Definitive Endoderm Kit (StemCell Technologies) for 3 days (day 0-3) and after the endoderm-induction stage, cells were dissociated with gentle cell dissociation reagent and passaged into 6-well dishes pre-coated with growth factor reduced Matrigel in ‘‘DS/SB’’ anteriorization media, consisting of complete serum-free differentiation media (cSFDM) base as previously described (Jacob et al., Cell Stem Cell 2017; Jacob et al., Nat Protoc 2019), supplemented with 2 µM Dorsomorphin (‘‘DS’’; Stemgent) and 10 µM SB431542 (‘‘SB’’; Tocris). For the first 24 hour after passaging, 10 µM Y-27632 was added to the media. After anteriorization in DS/SB media for 3 days (day 3-6), cells were cultured in ‘‘CBRa’’ lung progenitor-induction media until day 15, as we have previously published (Jacob et al., Cell Stem Cell 2017). ‘‘CBRa’’ media consists of cSFDM base supplemented with 3 µM CHIR99021 (Tocris), 10 ng/ml recombinant human BMP4 (rhBMP4, R&D Systems), and 100 nM retinoic acid (RA, Sigma). On day 15 of differentiation, live cells were sorted on a high-speed cell sorter (MoFlo Legacy or MoFlo Astrios EQ) to isolate NKX2-1+ lung progenitors based on CD47hi/CD26neg gating (Hawkins et al., JCI 2017). Sorted lung progenitors were resuspended in undiluted growth factor reduced 3D Matrigel (Corning) at a concentration of approximately 400 cells/µl and distal/alveolar differentiation of cells was performed in CK+DCI media, consisting of cSFDM base supplemented with 3 µM CHIR99021, 10 ng/mL rhKGF (CK), and 50 nM dexamethasone (Sigma), 0.1 mM 8-Bromoadenosine 30,50-cyclic monophosphate sodium salt (Sigma) and 0.1 mM 3-Isobutyl-1-methylxanthine (IBMX; Sigma) (DCI).

Extracted molecule

polyA RNA

Extraction protocol

Qiagen RNeasy Kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

Total RNA with ribosomal reduction

Data processing

Reads were mapped to GRCh38 with TdTomato reporter
Counts per gene were summarized using the featureCounts function from the subread package
edgeR package was used to import and filter the counts
Differential expression using MAST models
The limma package with its voom method, namely, linear modelling and empirical Bayes moderation was used to test differential expression (moderate t-test). P-values were adjusted for multiple testing using Benjamini-Hochberg correction (false discovery rate-adjusted p-value; FDR). Differentially expressed genes for each comparison were visualized using Glimma package, and FDR<0.05 was set as the threshold for determining significant differential gene expression. Enrichment analyses were carried out using Fgsea package.
Assembly: GRCh38 with TdTomato reporter
Supplementary files format and content: Tab separated file having raw read count for each sample in singular file

Submission date

Jan 17, 2023

Last update date

Sep 20, 2023

Contact name

Pushpinder Bawa

E-mail(s)

bpushpin@bu.edu

Organization name

Center for Regenerative Medicine

Department

Boston University Medical Campus