Project description:We obtained human embryonic and fetal lungs from 5-22 pcw for scRNAseq and scATACseq analysis. To focus on epithelial differentiation and region specialization, we deeply sampled 15, 18, 20 and 22 pcw lungs and separated proximal and distal regions while leaving lungs at 5, 6, 9 and 11 pcw intact. These cell samples (except for one at 6pcw) were split and processed for both scRNAseq and scATACseq.
Project description:Here, we used joint single-nuclei RNA-sequencing (snRNA-seq) and single-nuclei ATAC sequencing (scATAC) to profile freshly isolated crypts from the human fetal intestine and matched intestinal epithelial only organoids (also known as enteroids) derived from these crypts after one passage of in vitro growth. Organoids were grown in the standard 25% LWRN media with either 100 ng/ml of EGF or 1 ng/ml of EREG added. Fresh crypts were not placed in culture but rather immediately frozen for multiomic processing.
Project description:Individual TFs were overexpressed in fetal lung tip organoids from a doxycycline-inducible construct for 3 days, and organoids were maintained in the self-renewing (tip cell-promoting) medium throughout to rigorously assay the lineage-determining competence of the TF, followed by scRNA-seq. ASCL1, NEUROD1, and NEUROG3 were selected as key neuroendocrine regulators. We also selected the GHRL+ NE-specific RFX6 and NKX2.2, the pan-NE PROX1, and, as controls, the basal cell-specific TFs DeltaNTP63, TFAP2A, PAX9, and mNeonGreen-3xNLS.
Project description:Fetal lung samples at 12–20 post conception week (pcw) from the HDBR, up to 0.5cm3 in size, were embedded in OCT and flash-frozen in dry-ice cooled isopentane. Twelve-micron cryosections were cut onto Visium slides, haematoxylin and eosin stained and imaged at 20X magnification on a Hamamatsu Nanozoomer 2.0 HT Brightfield. These were then further processed according to the 10X Genomics Visium protocol, using a permeabilization time of 18 min for 12–17 pcw samples and 24 min for 19 pcw and older samples. Images were exported as tiled tiffs for analysis. Dual-indexed libraries were prepared as in the 10X Genomics protocol, pooled at 2.25 nM and sequenced in 4 samples per Illumina Novaseq SP flow cell with read lengths of 28 bp for R1, 10 bp for i7 index, 10 bp for i5 index, 90 bp for R2.
Project description:We obtained human embryonic and fetal lungs from 5-22 pcw for scRNAseq and scATACseq analysis. To focus on epithelial differentiation and region specialization, we deeply sampled 15, 18, 20 and 22 pcw lungs and separated proximal and distal regions while leaving lungs at 5, 6, 9 and 11 pcw intact. These cell samples (except for one at 6pcw) were split and processed for both scRNAseq and scATACseq.
Project description:We describe the NucleoATAC algorithm for high-resolution nucleosome positioning and occupancy determination using ATAC-seq. ATAC-seq was performed on Saccharomyces cerevisiae and Schizosaccaromyces pombe. Nucleosomes were called for both species as well as for the human GM12878 cell line using data from GSE47753. Additionally, ATAC-seq was performed on S. cerevisiae undergoing an osmotic stress time-course and nucleosomes were called for each time point.