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Sample GSM5685625 Query DataSets for GSM5685625
Status Public on Apr 19, 2022
Title B cell Nfil3-KO rep1
Sample type SRA
 
Source name spleen B cell
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: spleen
cell type: B cell
genotype: Nfil3-/-
treatment: IL-4 (20ng/mL) + LPS (5ug/mL) for 24 hours
Treatment protocol B cells were stimulated with IL-4 (20 ng/mL) and LPS (5 mg/mL) for 24 hours.
Growth protocol CD317- B220- MHC II+ CD11c+ XCR1+ CD172a- cDC1s were sorted from CD11c enriched splenocytes. CD19+ B220+ B cells were sorted from splenocytes.
Extracted molecule total RNA
Extraction protocol RNA was isolated from 200,000 live B cells sorted after stimulation or 120,000 cDC1 using NucleoSpin RNA XS Kit (Macherey-Nagel).
Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read.
RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a.
Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p5. Isoform expression of known Ensembl transcripts were estimated with Salmon version 0.8.2.
All gene counts were then imported into the R/Bioconductor package EdgeR and TMM normalization size factors were calculated to adjust for samples for differences in library size. Ribosomal genes and genes not expressed in the smallest group size minus one samples greater than one count-per-million were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into the R/Bioconductor package Limma. Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples with the voomWithQualityWeights. The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Differential expression analysis was then performed to analyze for differences between conditions and the results were filtered for only those genes with Benjamini-Hochberg false-discovery rate adjusted p-values less than or equal to 0.05.
Genome_build: Mouse Ensembl GRCm38.76 (mm10)
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Matrix table with merged differential expression results
 
Submission date Nov 10, 2021
Last update date Apr 19, 2022
Contact name Tiantian Liu
E-mail(s) ltt0321@gmail.com
Organization name Washington University in St. Louis
Department Pathology and Immunology
Lab Dr. Kenneth Murphy
Street address 660 S. Euclid Ave.
City Saint Louis
State/province MO
ZIP/Postal code 63110
Country USA
 
Platform ID GPL24247
Series (2)
GSE188564 Ablation of cDC2 specification by triple mutations in the Zeb2 enhancer [RNA-seq]
GSE188579 Ablation of cDC2 development by triple mutations within the Zeb2 enhancer
Relations
BioSample SAMN23036337
SRA SRX13097349

Supplementary file Size Download File type/resource
GSM5685625_9_b-ko.AATGCACCAA-ACACGCTCCT.gene_counts.txt.gz 2.7 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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