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Sample GSM4007045

Query DataSets for GSM4007045

Status

Public on Aug 18, 2020

Title

ODE_Attenuated_TGFb_rep1

Sample type

SRA

Source name

ODE_Attenuated_TGFb_rep1

Organism

Bos taurus

Characteristics

cell type: Ode macrophages

Growth protocol

Bovine BL3, TBL3, BL20, TBL20 B lymphocytes, and Ode macrophages were cultured in RPMI 1640 medium supplemented with 2 mM of L-glutamine (Lonza, catalogue number 12-702F) and 10 mM Hepes (Lonza, catalogue number 17-737E), 10 % heat-inactivated FBS (Gibco, catalogue number 10082147), 100 units/ml of Penicillin and 100 μg/ml of streptomycin (Lonza, catalogue number 17-602E) and 10mM beta-mercaptoethanol (Sigma-Aldrich, catalogue number M6250) for BL3/TBL3 and BL20/TBL20. The virulent (Vir) hyper-disseminating Ode cell line was used at low passage (53-71), while its attenuated (Att) poorly disseminating vaccine counterpart corresponded to passages 309-317. The OCI-LY19 cell line (DSMZ, ACC 528) was cultured in Minimum Essential Medium Eagle - Alpha Modification (Gibco, catalog number 12000063) supplemented with 2.2g/L of sodium bicarbonate (Thermofisher Scientific, catalog number 25080094), 20% FBS, 10 mM Hepes and 100 units/ml of Penicillin and 100 μg/ml of streptomycin. The RI-1 cell line (DSMZ, ACC 585) was cultured in RPMI1640 and supplemented with 10% FBS, 100 units/ml of Penicillin and 100 μg/ml of streptomycin and 10 mM Hepes. All cell lines were incubated at 37°C with 5% CO2 and were regularly tested for mycoplasma contamination.

Extracted molecule

total RNA

Extraction protocol

Cells were seeded in 3 biological replicates at a density of 2.5x105 cell/ml. RNA extraction was performed using the PureLink RNA Mini Kit (Life technologies, catalogue number 12183018A) following the manufacturer’s instructions. Briefly, cells were pelleted, lysed and homogenized using a 21-gauge needle, then 70% ethanol was added to cell lysates and samples loaded onto spin cartridges to bind RNA. After 3 washes RNA was eluted in RNase-free water. The quality of extracted RNA was verified using a Bioanalyzer 2100 and quantification carried using Qubit (Invitrogen, catalogue number Q10210).
Strand-specific RNA-sequencing (ssRNA-seq) libraries were prepared using the Illumina Truseq Stranded mRNA Sample Preparation Kit (Illumina, catalogue number RS-122-2101) following the manufacturer’s instructions. Briefly, 1ug of total RNA was used to purify mRNA using poly-T oligo-attached magnetic beads. mRNA was then fragmented and cDNA was synthesized using SuperScript III reverse transcriptase (Thermofisher, catalogue number 18080044), followed by adenylation of the 3’-end, barcoding and adapter ligation. The adapter ligated cDNA fragments were then enriched and cleaned with Agencourt Ampure XP beads (Agencourt, catalogue number A63880). Libraries validation was conducted using the 1000 DNA kit on 2100 Bioanalyzer (Agilent Technologies, catalogue number 5067-1504) and quantified using qubit (Thermofisher, catalogue number Q32850). ssRNA libraries were sequenced on Illumina Hiseq2000 and Hiseq4000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

bcl2fastq2 Conversion Software
Trimming of Illumina adaptor sequences and low quality reads were done using Trimmomatic (v 0.33)
The strand-specific reads were mapped on to Bovine genome (bosTau7; Btau_4.6.1; GCF_000003205.5) using Tophat2 (-g 1 --library-type fr-firststrand). The samples with respective replicates were analyzed further for differential gene expression by three different tools, baySeq, DESeq2 (fitType ="local") and CuffDiff2 with default parameters unless mentioned specifically. The count values for DESeq2 and baySeq were calculated from BAM files using HTSeq-count tool. The transcriptome quality plots were generated by cummeRbund package (v2.14.0).
Genome_build: Bos taurus4.6.1 (bosTau7)

Submission date

Aug 05, 2019

Last update date

Aug 18, 2020

Contact name

AMIT KUMAR SUBUDHI

E-mail(s)

amit.subudhi@kaust.edu.sa

Phone

+96540375986

Organization name

King Abdullah University of Science and Technology