|
| Status |
Public on May 05, 2020 |
| Title |
Gfi1_stroma-free_Con_IP_2 |
| Sample type |
SRA |
| |
|
| Source name |
Cultured utricles
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Cultured utricles
genotype: Gfi1-Cre; Rpl22tm1.1Psam/J
condition: Control; 37 degrees C culture
sample type: ribotag immunoprecipitated
|
| Treatment protocol |
Cultured utricles were then either exposed to heat shock in microcentrifuge tubes placed in a 43°C in a water bath for 30 minutes and allowed to recover for 2 hours, or they remained at 37°C under control culture conditions.
|
| Growth protocol |
Mice were euthanized by CO2 asphyxiation followed by decapitation. Utricles were immediately dissected in M199 media and the epithelial roof was gently dissected away, leaving the otoconia intact. The utricles were incubated overnight at 37°C (95% air/5% CO2) in DMEM/F-12 media with 5% FBS , and 50 U/mL penicillin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Micro Plus kit (Qiagen) including the genomic DNA removal spin column step.
Sequencing libraries were prepared using the SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA). Dual indexed libraries were prepared using the Nextera XT DNA Library Preparation kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
underlying stroma removed
Raw read counts
|
| Data processing |
Map to mouse genome using STAR/2.5.2a
Command line parameters STAR --genomeDir /data/GCBCNIDCR/Refs/Stargenomes/MOUSE-GRCm38.vM11c/ --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMstrandField None --outFilterType BySJout --outFilterMultimapNmax 20 --outFilterMatchNminOverLread 0.25 --outFilterScoreMinOverLread 0.25 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.3 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --readFilesIn /data/GCBCNIDCR/RNASeqProjects/17_012Ribotag/input/Gfi1_Con_input_1_R1_all.fastq.gz /data/GCBCNIDCR/RNASeqProjects/17_012Ribotag/input/Gfi1_Con_input_1_R2_all.fastq.gz --readFilesCommand zcat --runThreadN 56 --outFileNamePrefix Gfi1_Con_input_1.star. --outSAMtype BAM SortedByCoordinate --outSAMattrRGline ID:Gfi1_Con_input_1 LB:library PL:ILLUMINA PU:machine SM:Gfi1_Con_input_1 CN:NIDCD_GCBC --bamRemoveDuplicatesType UniqueIdentical --quantMode GeneCounts --sjdbGTFfile /data/GCBCNIDCR/Refs/Stargenomes/MOUSE-GRCm38.vM11c/GRCm38.vM11.annotation.erccTgSIRVs.gtf --outSAMunmapped Within --outWigType None --outWigStrand Stranded
ReadsPerGene.out.tab file generated by STAR: Column1=geneID; Column2=counts unstranded RNA-seq; Column3=counts for 1st read strand aligned with RNA; Column4=counts for 2nd strand aligned with RNA
Raw gene counts were analyzed for differential gene expression using the RNA-Seq statistical method DESeq2 (Love et al., 2014).
Genes significantly enriched in the IP versus the matching input sample (log2>1 and adjP<0.05) were considered for further analysis in comparison of Control versus Heat Shock experiments.
For each Cre-driver experiment, expression values of the enriched genes (input versus IP) were compared between control and heat shocked samples. Significantly different genes could be either up or down-regulated and adjP-values <0.05.
Genome_build: GRCm38.vM11
Supplementary_files_format_and_content: raw read counts
|
| |
|
| Submission date |
Oct 30, 2019 |
| Last update date |
Nov 21, 2022 |
| Contact name |
Robert Morell |
| E-mail(s) |
[email protected]
|
| Organization name |
National Institute on Deafness and Other Communication Disorders
|
| Street address |
35A Convent Drive, Room 1F-103
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL18480 |
| Series (1) |
| GSE139593 |
Cell Specific Transcriptional Responses to Heat Shock in the Mouse Utricle Epithelium |
|
| Relations |
| BioSample |
SAMN13158002 |
| SRA |
SRX7071958 |
