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Sample GSM4041755 Query DataSets for GSM4041755
Status Public on Oct 08, 2019
Title SC_PBS_2
Sample type SRA
 
Source name Lung Innate lymphoid cells
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: lung
cell type: Innate lymphoid cells
batch: D
cgrp-treated: No
il33-treated: No
timepoint: 3 days
in vitro vs. in vivo: In vivo
protocol: ScRNA-seq (10x)
Treatment protocol Nasal administration of PBS, CGRP, IL-33 or IL-33+CGRP daily for three consecutive days. One day after the last treatment, lung ILCs were isolated by fluorescence-activated cell sorting.
Extracted molecule polyA RNA
Extraction protocol Lungs were digested with the lung dissociation kit (Miltenyi Biotec) and subsequently enriched for CD90.2+ cells using CD90.2 MicroBeads (Miltenyi Biotec). Lung ILCs were isolated by fluorescence-activated cell sorting as 7AAD- CD45+ CD90.2+ CD127+ Lineage (CD3, CD4, CD8, CD11b, CD11c, CD19, NK1.1, TCRβ, TCRγδ)- cells.
Sorted ILCs were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3′ Reagent Kits v3 (1000075) according to manufacturer’s protocol (10x Genomics). The generated scRNA-seq libraries were sequenced on a HiSeq using a read structure of 28 bp for Read 1, 8 bp for Index 1, and 96 bp for Read 2.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
 
Description PBS_2.filtered_feature_bc_matrix.h5
Data processing For bulk RNA-seq (in vitro samples) data, the raw data was converted to fastq files using bcl2fastq 2.17.1.14 with options “--minimum-trimmed-read-length 10 --mask-short-adapter-reads 10”. Transcript quantification was done using Kallisto 0.42.3 (Bray et al., 2016) with the mm10 mouse genome annotation, and transcript counts were then converted to gene counts and normalized TPM values using the R package tximport (Soneson et al., 2015).
For scRNA-seq data data, gene counts were obtained by aligning reads to the mm10 genome using CellRanger software (v1.3) (10x Genomics). To remove doublets and poor-quality cells, cells were excluded from subsequent analysis if they were outliers in their sample or condition of origin in terms of number of genes or number of unique molecular identifiers (UMIs), which left 83.1% and 84.2% of cells from CGRP and PBS, respectively, and 90.3% and 93% of cells from IL-33 and IL-33+CGRP conditions. Sample-specific minimum and maxium cut-offs per cell were 900–5,000 genes for IL-33 and IL-33+CGRP conditions and 900–3,900 genes for CGRP and PBS. Another 2.1% of the remaining cells were excluded for having greather than 10% of mitochondrial gene counts.
For scRNA-seq data, normalization of gene counts was performed using regularized negative binomial regression via the SCTransform() function from Seurat v3, with the “batch_var” parameter set to the replicate indicator variable and the “vars_to_regress” parameter set to the variable capturing the percentage of mitochondrial gene counts in each cell (Butler et al., 2018; Hafemeister and Satija, 2019). This step takes the place of the typical steps of log-normalization, variable gene selection, and scaling. We used log1p of the corrected counts to compute principle components analysis (PCA).
ATAC-seq: Read alignment, filtering, visualization of signal tracks, and measurement of quality control metrics was performed using a publicly available ATAC-seq pipeline (Lee et al., 2016). Briefly, reads were aligned to the mm10 genome using Bowtie2 and filtered to remove duplicates and mitochondrial reads. Alignment files were merged for biological replicates for peak-calling using MACS2 (Zhang et al., 2008). Read counts per peak were compiled using the bedtools multicov tool. Counts were normalized and processed for differential peak accessibility between conditions using DESeq2 (Love et al., 2014). Gene annotation with putative regulatory elements was performed using GREAT with default parameters (McLean et al., 2010).
Genome_build: mm10
 
Submission date Aug 21, 2019
Last update date Oct 08, 2019
Contact name Samantha Riesenfeld
Organization name Broad Institute
Lab Regev
Street address 415 Main St
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
 
Platform ID GPL21273
Series (1)
GSE136154 Calcitonin gene related peptide negatively regulates alarmin-driven type 2 innate lymphoid cell responses
Relations
BioSample SAMN12617273
SRA SRX6749262

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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