title: Illumina HiSeq 2000 paired end sequencing; Comprehensive RNA-Seq Profiling of the Lung Transcriptome of Bashbay Sheep in Response to Experimental Mycoplasma ovipneumoniae Infection
design: Comprehensive RNA-Seq Profiling of the Lung Transcriptome of Bashbay Sheep in Response to Experimental Mycoplasma ovipneumoniae Infection
library_construction_protocol: The experimental procedures were approved by the Animal Welfare Committee of Animal Science and Technology Institute, Shihezi University. Nine Bashbay sheep were procured from the farm of breeding sheep in Yumin county, Xinjiang. All the lambs were 2- to 3-months-old and weighed 10–15 kg, and were also confirmed to be sero-negative for MO using a commercial ELISA kit (R&D, USA).The sheep were respectively slaughtered in each group(three sheep in each group) before the infection (0d group), 4d after infection (4d group), and 14d after infection (14d group). The lung samples were collected and immediately frozen in liquid nitrogen and transferred to -80 °C.  After the sheep were corona in the slaughterhouse, they were sacrificed by bloodletting from the neck, and the sheep were confirmed to be dissected after dying. The whole process was supervised by members of the Animal Welfare Committee of the College of Animal Science and Technology of Shihezi University. The experimental sheep were purchased from the sheep farm in Yumin County, Xinjiang. The sheep were kept in grazing and fed freely. The lamb was not invaded during its growth. The sheep were transported to the Experimental Animal Breeding Center of Shihezi University for adaptive breeding one week before treatment. The experimental group was then taken out under the same feeding conditions for challenge and isolation. The sheep were free to eat during the treatment and were not invaded. The experimental procedure was approved and supervised by the Animal Welfare Committee of the College of Animal Science and Technology of Shihezi University. The raw image data files sequenced by high-throughput sequencing (such as the sequencing platform such as illumina HiSeqTM2500) are converted into Sequenced Reads by Base Calling analysis. We call it Raw Data or Raw Reads, and the result is FASTQ. (abbreviated as fq) file format storage, which contains sequence information of the sequencing sequence and its corresponding sequencing quality information. The first line begins with \"@\" followed by the Illumina Sequence Identifiers and descriptive text (selective part); the second line is the base sequence; the third line begins with \"+\" followed by illumina The sequencing identifier (selective portion); the fourth row is the sequencing quality of the corresponding sequence (Cock et al.). The ASCII value corresponding to each character in the fourth line is subtracted by 33, which is the sequencing quality value corresponding to the second row of bases. If the sequencing error rate is expressed as e and the base mass value of the illumina HiSeqTM 2500 is expressed in Qphred, the following relationship is available: Qphred = -10log10(e). MirVana mRNA isolation kit (Ambion-1561, USA) was used to isolate total RNA in accordance with the manufacturer's instructions. Total RNA was purified with Agencourt AMPure XP (Beckman Coulter). The purity of RNA was assessed by electrophoresis on 1% agarose gel The concentration and integrity of RNA were determined using NanoDrop 2000 (Thermo Scientific) and Agilent Bioanalyzer 2100 (Agilent Technologies), respectively. The samples with an RNA Integrity Number (RIN) ≥ 7 were used for subsequent analysis. Four microgram of RNA was used for the synthesis of cDNA, which was used for the construction of libraries with TruSeq Stranded mRNA LTSample Prep Kit (Illumina, USA) according to the manufacturer's recommendations.