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Sample GSM7506625

Query DataSets for GSM7506625

Status

Public on Dec 27, 2023

Title

LTT 1 (PA 8695)

Sample type

SRA

Source name

CD34-CD105+CD117+bRBC+ FACS-purified bone marrow cells

Organism

Papio anubis

Characteristics

tissue: CD34-CD105+CD117+bRBC+ FACS-purified bone marrow cells
treatment: Long-term (>265d) RN-1 treatment (PA 8695)

Treatment protocol

CD34-CD105+CD117+bRBC+ bone marrow cells were FACS-purified from 2 untreated contrOl baboons (CTRL1 and CTRL2, PA 8697 and PA8548, two baboons (STT1 and STT2; PA 8697 and 8548) treated for three days with RN-1 (0.25mg/kg/d) and two baboons (Ltt1 and Ltt2, PA 8695 and PA 8698) treated with RN-1 (0.25mg/kg/d; 5d/wk) for >265 days.

Extracted molecule

total RNA

Extraction protocol

RNA was isolated using the RNeasy minikit (Qiagen #74104)) and treated with Rnase-free Dnase (Ambion #AM1906) according to the manufacturer's insructions.
Sequencing libraries were prepared using strand-specific QuantSeq 3’ RNAseq kit (Lexogen, FWD, Cat. No. 015). The QuantSeq protocol generates only one fragment per transcript, resulting in accurate gene expression values, and the sequences obtained are close to the 3’ end of the transcripts. Total RNA in the amount of 50 nanograms per sample was used as an input. Library construction was performed according to the manufacturer’s protocol. In brief, during the first strand cDNA synthesis, an oligo dT primer containing an Illumina-compatible sequence at its 5’ end was hybridized to mRNA, and reverse transcription was performed. After that, the RNA template was degraded, and during second strand synthesis, the library was converted to dsDNA. Second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5’ end. The double-stranded libraries were purified by using magnetic beads to remove all reaction components. Next, the libraries were amplified to add the complete adapter sequences required for cluster generation and to generate sufficient material for quality control and sequencing. A number of PCR amplification cycles was 18, as determined by test qPCR using a small pre-amplification library aliquot for each individual sample. Final amplified libraries were purified, quantified, and fragment sizes were confirmed to be within 275-300 bp by gel electrophoresis using Agilent 4200 TapeStation (D5000 Screen Tape). Concentration of the final library pool was confirmed by qPCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw reads were aligned to reference genome Panu_2.0 using BWA MEM.39 Gene expression was quantified using FeatureCounts.40 Differential expression statistics (fold-change and p-value) were computed using edgeR,41,42 including both multi-group comparisons using generalized linear models as well as pair-wise tests between experimental groups using the exactTest function. In all cases we adjusted p-values for multiple testing using the false discovery rate (FDR) correction of Benjamini and Hochberg.43
For STT vs CTRL, baboon1 (PA 8697) and baboon2 (PA 8548) are involved. In this case, we can
used ANOVA to compare the treatment effect as well as the baboon effect.
For LTT vs CTRL, all four baboons are involved in the analysis. We
simply compared LTT (PA8695 and PA 8698) vs CTRL (Pa 8697 and PA 8548) without considering baboon effect (which can't
Assembly: be identified by this experiment design).
Supplementary files format and content: Table submitted (STT vs CTRL DEG Pathway.xlsx). This table contains data showing comparison of pooled CTRL1 and CTRL2 with STT1 and STT2)
Supplementary files format and content: Table submitted (LTT vs CTRL DEG Pathway.xlsx). This table contains data showing comparison of pooled CTRL1 and CTRL2 with LTT1 and LTT2)

Submission date

Jun 22, 2023

Last update date

Dec 27, 2023

Contact name

Donald Lavelle

E-mail(s)

dlavelle@uic.edu

Phone

8473630165

Organization name

University of Illinois at Chicago