title: GSM4200581: control-60 rep4; Bos taurus; RNA-Seq
library_construction_protocol: Total RNA was extracted in oocytes of vehicle (n = 5) and bacteria (n = 3) infused heifers at day 4, and vehicle (n = 6) and bacteria (n = 3) infused heifers at day 60. Oocytes were thawed and suspended in 350 μL RLT buffer for the extraction of total RNA using the RNeasy Micro kit (Qiagen) according to the manufacturer's instructions. Total RNA concentration was determined using a Qubit 2.0 Fluorometer (Fisher Scientific) and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA). To produce RNAseq libraries for sequencing, 200 pg of total RNA was used for library construction using SMARTer Universal Low input RNA kit (Takara Bio, Mountain View, CA), combined with Illumina Nextera DNA Library Preparation Kit (Illumina, San Diego, CA), according to the manufacturer's instructions. Briefly, first strand cDNA was primed by a modified N6 primer (the SMART N6 CDS primer), then base-paired with additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting in single-stranded cDNA containing sequences that are complementary to the SMARTer oligonucleotide. The SMARTer anchor sequence and the N6 sequence then served as universal priming sites for DNA amplification by PCR for 10 cycles. A total of 125 pg of cDNA was then fragmented by a tagementation reaction and adapter sequences were added to the template cDNA by PCR amplification.
attributes.tag: GEO Accession
attributes.value: GSM4200581