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Sample GSM7445938

Query DataSets for GSM7445938

Status

Public on Dec 27, 2023

Title

H504

Sample type

SRA

Source name

hip cartilage tissue, 50mPa

Organism

Mus musculus

Characteristics

tissue: hip cartilage
strain: C57/BL6
Sex: male
library type: unstranded

Treatment protocol

Femoral heads were isolated from 4-week-old male C57/Bl6 mice. Embyronic metatarsals were isolated from E15 embryos of C57/Bl6 mice. After 24 hours of culture, hips caps and metatarsals were placed into the hydrostatic pressure vessel and subjected to 0mPa (control), 5mPa (physiological) or 50mPa (injurious) hydrostatic pressure for 1 hour. Following mechanical stimulation, tissues were placed back into the incubator and cultured for a further 24 hours in the respective media

Extracted molecule

total RNA

Extraction protocol

The RNA was extracted from hip and metatarsal cartilage tissues using the following protocol: after collection tissue samples were pooled (n=6 metatarsals and n=2 hip caps for each sample), transferred to eppendorf tubes and frozen at -80ºC. Tissue (<100mg) were defrosted on ice and 1ml Trizol was added to each sample; tissues were homogenised making sure to keep them cool by putting on ice every 15secs. Samples were Incubated at room temperature for a minimum of 10 minutes to allow for cell lysis and centrifuged at 12,000 g for 15 minutes at 4 °C to pellet the excess tissue, whilst retaining RNA in solution. The supernatant was transferred to a clean tube and 200 μL of chloroform added. After vigorous shaking for 20 seconds the samples were incubated at room temperature for 3 minutes and then centrifuged at 12,000 g for 15 minutes at 4 °C to enable phase separation. The upper, aqueous phase was transferred to a new eppendorf avoiding the Interface. 1 volume of 70% ethanol was added and the samples were mixed thoroughly by vortexing. 700 μl of the sample was transferred to an RNeasy Mini spin column (Qiagen). From this point onwards the manufacturer's recommendation were followed. The elution was done in 30μl of RNase free water and repeated twice with the first eluate. The concentration and purity of the RNA samples were assessed by measurments on the Nanodrop One C spectrophotometer (Labtech) and the quality of the RNA was assessed on a TapeSation 4200 (Agilent Technologies).
The libray construction kit was the Universal Plus Total RNA-seq with Nuquant (Tecan Genomics) which produces stranded RNAseq libraries with Illumina compatible adaptors and unique dual indexes.Input total RNA was between 20 and 200 ng and the amplification cycles were optimized by QPCR based on the manufacturer's recommendations. The cycles of amplifications varied based on the optimization by QPCR on 1/10 of the available volume before amplificatioj and were determined so that the chosen number of cycles were in the middle exponential PCR amplification curve.The procedure followed was as specified by the manufacturer's recommendation except for a technical error made during library preparation which affected the strandness of the following samples HC3, H501, H502, H503, H504, M504. Those samples were analysed using the unstranded library analysis pipeline while the reminder of the samples were analysed using the stranded library analysis pipeline :HC1, HC2, H51, H52, H53, H54, MC1, MC2, MC3, MC4, M501, M502, M503. Samples H502 and HC4 were ultimately identified as outliers and were eliminated from the final data analysis.
RNA seq.The RNA samples had a RINe value which varied from low to medium (1.5-7).DV200 quality metric (the percentage of RNA fragments above 200 nt) which is part the TapeStation 4200 software was used to assess the quality of partially degraded RNA and the analysis showed a range between 53.51-87.4%. Given the variable range of RNA sample quality the library construction strategy exploited the depletion of ribosomal RNA rather than the purification of mRNAs from total RNA.The probe mix for mouse and the Universal Plus any deplete module used was part of the library construction kit that was used to prepare the RNAseq libraries: Universal Plus Total RNA kit with NuQuant (Tecan Genomics)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Sequenced reads were trimmed for adaptor sequences and for quality (minimum 20) using trim-galore v0.6.5.
Trimmed reads were quantified using kallisto quant
kallisto quantifications were converted to gene level by tximport, then normalised and analysed for differential expression using DESeq2
Assembly: GRCm39
Supplementary files format and content: Tab-delimited text file. Matrix table with raw gene counts for every gene and every sample, generated by tximport

Submission date

Jun 05, 2023

Last update date

Dec 27, 2023

Contact name

Katherine Ann Staines

E-mail(s)

k.staines@brighton.ac.uk

Organization name

University of Brighton

Street address

Lewes Road