title: GSM4491952: CON_8923_30D_Mammary [8923_30D_R_CTGCTTCC-GATAGATC]; Bos taurus; RNA-Seq
library_construction_protocol: After collection samples were snap frozen in liquid nitrogen, and stored at -80C until extraction. Tissue was weighted (~ 0.05 g), immediately placed in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) (1  mL) and homogenized using a Mini-Beadbeater-24 (Biospec Products Inc., Bartlesville, OK) with two 30 s cycles, and 1 min incubation on ice in between the cycles. Samples were then centrifuged for 10 min at 12,000 × g and 4 °C, and the supernatant was transferred to a separate tube and mixed with chloroform (0.2 mL). After centrifugation for 15 min at 12,000 × g and 4 °C, the aqueous phase was transferred to a new tube, mixed with 100% ethanol (0.75 mL), and total RNA was cleaned using miRNeasy mini kit columns (Qiagen, Hilden, Germany) following manufacturer's protocols. During purification, genomic DNA was removed using the RNase-Free DNase Set (Qiagen, Hilden, Germany). Quantity was determined using a Qubit Fluorometer (Invitrogen, Carlsbad, CA), while integrity was assessed via a Fragment Analyzer™ (Agilent Technologies, Santa Clara, CA). All samples had an RQN score greater than 7.0. RNA samples were stored at −80 °C until analysis. After quality control, samples were handed to the DNA Service Lab of the University of Illinois Roy J. Carver Biotechnology Center for library preparation and sequencing. RNAseq libraries were constructed with the TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA). The final libraries were quantitated with a Qubit Fluorometer (Invitrogen, Carlsbad, CA) and the average library fragment length was determined on a Fragment Analyzer™ (Agilent Technologies, Santa Clara, CA). The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad, Hercules, CA) for accurate pooling of the barcoded libraries and maximization of number of clusters in the flowcell.
identifiers.namespace: GEO
identifiers.id: GSM4491952