GEO help: Mouse over screen elements for information.
|Public on Nov 22, 2016
|BrdU was injected at a concentration of 50mg/kg body weight at 12 and 2 hours before sacrifice. Sorted epithelial cells were fixed and stained using the FITC BrdU Flow Kit (559619, BD Biosciences). Cells were analyzed on an LSRFortessa (BD Biosciences) flow cytometer. After 7 days in vitro, cultures were treated with 5U/mL dispase in Ham’s F12 and incubate at 37°C for 30 minutes. Halfway through incubation, cultures were disrupted by pipetting. Cells were collected and resuspended in 0.05% trypsin and incubated at 37°C for 30 minutes. Samples were vortexed several times during incubation. Cells were then filtered through a 70μm filter stained with 7AAD and sorted for GFP+7AAD-. One thousand sorted epithelial cells were cultured with one hundred thousand mouse lung fibroblasts in 50% Matrigel as described previously.
|Five thousand flow sorted epithelial cells were cultured with one hundred thousand mouse lung fibroblasts and Matrigel (BD Biosciences) seeded onto Transwell filter inserts (BD Biosciences) as previously described (Chen et al., 2012). Briefly, the epithelial/fibroblast mixture was added to an equal volume of growth factor reduced Matrigel (BD Biosciences) and seeded to the apical surface of 24-well transwell filter inserts (BD Biosciences) placed in 24-well flat-bottom culture plates. The solution was allowed to polymerize for 30 min at 37°C, then basic medium was added to the basal compartment of the well. Cell cultures were maintained for 7-14 days at 37ºC in a humidified incubator (5% CO2). Colony-forming efficiency was calculated as the percentage of seeded cells that give rise to colonies, imaged on a Zeiss Axiovert40 fluorescent microscope and quantitated using FIJI.
Scgb1a1-CreERTM; Rosa26-mT/mG and Scgb1a1-CreERTM; Rosa26R-Confetti mice were previously described (Farin et al., 2015; PMID 25564721). These mice were crossed to p53flox (JAX stock number 008462), p53-/- (JAX stock number 002101), or Super p53 (kindly provided by David Kirsch) mice to generate experimental animals. Mice were injected with 3 x 200mg/kg, 1 x 250mg/kg, or 1 x 5mg/kg body weighttamoxifen in corn oil for high dose, RNA-Seq, and low dose experiments respectively. All mice were maintained and treatments were carried out according to IACUC approved protocols.
|Cell capture, lysis, reverse transcription and cDNA amplification was performed on the C1 IFC for mRNA-seq on Fluidigm C1 Single-Cell Auto Prep System following the manufacturer’s protocol. Medium sized C1 mRNA-Seq chips were used to capture each cell-cycle fraction. The C1 Auto-prep system captured the dissociated single cells across 96 wells and performed cell lysis, cDNA synthesis with reverse transcription and PCR reaction using the SMARTer Ultra Low Input RNA Kit. Cells captured across the 96 wells are manually inspected as a quality control measure to remove empty well, doublets or debris containing wells. 92 ERCC spike-ins of known concentration were added during the lysis step to assess and quantify technical variation. cDNA from several representative cells were checked by High Sensitivity DNA chips using Fragment Analyzer (Advanced Analytical).
Libraries for each of the 96 captured cells were prepared using the Illumina Nextera XT DNA sample preparation kit with 96 dual barcoded indices. Single cell libraries were multiplexed and sequenced across 4 lanes of a NextSeq 500 platform (Illumina) using 75 single-end sequencing. On average, about 6-7 million reads would be generated from each single cell library.
|Illumina NextSeq 500
|Raw reads obtained from RNA-Seq were aligned to the transcriptome using Bowtie (version 1.1.1) / RSEM (version 1.2.20) with default parameters
Expression counts for each gene in all samples were normalized by the total number of reads mapped to the transcriptome in order to account for sequencing depth (transcripts per million; TPM) with the correction of size factors derived from ERCC spike-in.
We imposed additional quality control criteria to remove low quality cells: 1), raw sequencing reads <1M; 2) among all sequencing reads, percentage of total mapped read is <50%; 3) among mapped reads, percentage of exonic reads <50% and 4) percentage of ERCC spike-in reads>40% to eliminating majority of the dying or dead cell.
The expression counts were transformed into log scale. To identify the subpopulation among the samples in an unsupervised way, principle component analysis (PCA) was performed to select most varied genes across all single cells based on their eigenvectors with FactoMineR v1.31 in R/Bioconductor v3.2. The top 250 most varied genes from PCA was clustered with Euclidean distance matrix with ggplots2 v1.0.1 in R/Bioconductor.
Genome_build: a custom reference containing both 92 ERCC sequences and the mouse transcriptome GRCm38 reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based on Release M3 annotation.
Supplementary_files_format_and_content: tab-delimited text files include tpm values for each Sample calculated by RSEM
|Feb 17, 2016
|Last update date
|Oct 11, 2021
|Cedars Sinai Medical Center
|8687 Melrose Ave, PDC B230
|p53 is a critical regulator of airway epithelial progenitor cell homeostasis.