title: GSM4162551: treatment-IC rep2 [401IC]; Bos taurus; RNA-Seq
library_construction_protocol: Two weeks prior to slaughter, the oestrous cycles of all the animals were synchronized using the modified 5-d Co-synch protocol described above. Animals were slaughtered by captive bolt and exsanguination 94 days after infusion. Reproductive tracts were collected, transported on ice, and processed within 1 h of slaughter. Endometrial tissue was dissected away from underlying myometrium, and oviduct samples were collected by extrusion into a collection vessel. Granulosa cells were isolated from dominant follicles > 8 mm diameter by aspiration, using a sterile 20-gauge needle and syringe containing collection medium (0.5% BSA, 20 mM HEPES, 2 mM sodium pyruvate, 10 IU/ml heparin, 100 U/ml penicillin, 100 ug/ml streptomycin in Medium 199); the aspirates were centrifuged at 500 × g to isolate the granulosa cells. Samples of caruncular and intercaruncular endometrium, oviduct isthmus and ampulla, and granulosa cells were snap frozen in liquid nitrogen and stored at -80°C. Technical problems prevented collection of the oviduct from one bacteria-infused animal and the intercaruncular endometrium from another bacteria-infused animal Samples were thawed, suspended in RLT buffer, and RNA extracted using the RNeasy Micro kit. To produce RNAseq libraries, 500 ng (endometrium and oviduct) or 200 ng (granulosa) total RNA was used for mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation Module, followed by RNA library construction with NEBNext Ultra II Directional Library Preparation (both New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. Briefly, extracted mRNA was fragmented in NEBNext first-strand synthesis buffer at 94°C, followed by first-strand cDNA synthesis using reverse transcriptase and random primers. Synthesis of double-stranded cDNA was performed using the second strand master mix provided with the kit. The resulting double-stranded cDNA was end-repaired, dA-tailed and ligated with NEBNext adaptors. Finally, libraries were enriched by amplification and purified by Meg-Bind RxnPure Plus beads (Omega Biotek, Norcross, GA, USA). Barcoded libraries were sized on a bioanalyzer, and quantified by Qubit and qPCR. All individual libraries were pooled, and equal molar concentrations were run on an Illumina HiSeq3000 sequencer. Both library construction and sequencing were performed at the ICBR of the University of Florida
attributes.tag: GEO Accession
attributes.value: GSM4162551