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| Status |
Public on Mar 31, 2022 |
| Title |
PKBuffer Brain PBS sample #2 [BB1P2] |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: Brain
tissue treatment: Phosphate Buffered Saline (PBS) treated
rna isolation buffer: PIXUL Proteinase K Buffer
mouse number: 5
genotype: Female 8-12 week-old C57bl/6
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| Treatment protocol |
Female 8–12-week-old C57bl/6 mice were injected intraperitoneally (IP) with 5mg/kg lipopolysaccharide (LPS) in 200ml PBS or with 200ml PBS as control. The specific protocol was approved by Institutional Animal Care and Use Committee (IACUC) at the University of Washington. After 12 hours mice were euthanized by isoflurane overdose and confirmatory cervical dislocation. Brains, hearts, kidneys, and livers were harvested, immediately placed on ice, and then frozen in CryoTrays
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| Extracted molecule |
total RNA |
| Extraction protocol |
1-2mg samples of frozen tissue were sampled with the CryoCore and put into separate wells of a 96 well plates. 100ul of either PIXUL PK Buffer or TRIZOL buffer wer added to the wells and then all samples were sonicated in PIXUL sonicator (Matchstick Technologies, Inc Kirkland WA and Active Motif Carlsbad, CA). Samples are processed for 30 seconds in PIXUL with settings: Pulse=50, PRF=1.0 Burst=20. TRIZOL samples then received additional 100ul of TRIZOL and followed manufacturers protocol for RNA isolation. PIXUL PK Buffer samples are immediately incubated (After sonication) in the same PIXUL plate at 95C for 20 minutes, and then put on ice for 5 minutes. Cooled samples are centrifuged in a plate centrifuge for 10 minutes at 4,200g and 4C and then put back on ice. 60ul of clear supernatant (without any floating residue) from each well is carefully transferred via pipette to a new semi-skirted 96 well PCR plate on ice. Isolation of nucleic acids with 1.8x SPRI beads is done as per the Omega Bio-tek protocol (Norcross, GA) with a two-minute final SPRI bead drying time and final elution to 50ul of ultrapure ddH2O and then put samples on ice. Finally, DNase I and RNAse Out are added to the eluted RNA samples and DNase I digestion is done as per the manufacturer’s protocol.
Sequencing libraries were prepared using Zymo-Seq RiboFree Total RNA Library Kit with starting RNA between 205-480ng and libraries amplified between 13-14 cycles of PCR as per manufacturers protocol. Libraries were diluted as per Illumina NextSeq2000 Sequencing System Guide to a final pooled loading concentration of 650pM in resuspension buffer (RSB) plus Tween 20 with a 10% PhiX spike-in and sequenced in Illumina P2 cartridges on NextSeq 2000 that employed a dual-index, paired-end, 75 base read length (PE75).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
2mg sample from frozen organ in CryoTray
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| Data processing |
Illumina RTA version 3.9.25
Index for alignment was built using the 'buildindex' function from the Bioconductor 'Rsubread' (version 2.3.2) package. Index was assembled from the NCBI Genome Reference Consortium Mouse Build 39 (GRCm39) RefSeq assembly
Reads were aligned and counted in RStudio using the Bioconductor ‘RSubread’ (version 2.8.2) package. Alignment was done with the ‘align’ function on the paired gzFASTQ files creating sorted BAM files. Align commands: type = "rna", input_format = "gzFASTQ", output_format = "BAM", sortReadsByCoordinates = TRUE, PE_orientation = "fr"
‘featureCounts’ function was used with the NCBI GRCm39 RefSeq Annotation and the commands: isGTFAnnotationFile=TRUE, countMultiMappingReads = FALSE, strandSpecific = 2, isPairedEnd=TRUE.
Assembly: mm39
Supplementary files format and content: tab deliminted text files include raw gene counts and gene annotation for each sample.
Supplementary files format and content: .csv table with raw gene counts for every gene and every sample
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| Submission date |
Mar 28, 2022 |
| Last update date |
Jul 28, 2022 |
| Contact name |
Karol Bomsztyk |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
UW Medicine South Lake Union
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| Lab |
Bomsztyk Lab
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| Street address |
850 Republican St
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL30172 |
| Series (1) |
| GSE199598 |
CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis |
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| Relations |
| BioSample |
SAMN27023740 |
| SRA |
SRX14637809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5975929_BBP2counts.txt.gz |
194.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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