title: GSM6012590: slick rep7; Bos taurus; RNA-Seq
library_construction_protocol: RNA extraction was performed with the Qiagen RNeasy Mini kit (Germantown MD, USA) according to the user's guide. Quantity of RNA was determined using the QUBIT fluorescent method (Invitrogen, Carlsbad, CA, USA) and Agilent Bioanalyzer (Santa Clara, CA, USA). An amount of 100 ng of high quality of total RNA with RIN of 7 or higher was used for library construction using the reagents provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA) and the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer's instructions. Briefly, 100 ng total RNA was used for mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module. Then the poly A enriched RNA was fragmented in NEBNext First Strand Synthesis Buffer via incubation at 94 °C for the desired time. Synthesis of ds cDNA was done using the 2nd strand master mix provided in the kit, followed by end-repair and dA-tailing.  At this point, Illumina adaptors were ligated to the sample. Finally, library was amplified and then purified with AMPure beads (Beckman Coulter, Brea, CA, USA).  Library size and mass were assessed by analysis in the Agilent TapeStation using a DNA5000 Screen Tape.  Quantitative PCR was used to validate the library's functionality using the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA) and monitoring on the BioRad CFX 96 real-time PCR system (Richmond, CA, USA). Twelve barcoded libraries were pooled equimolarly for sequencing simultaneously for 0.23 lanes using the Illumina NovaSeq6000. Normalized libraries were submitted to the Illumina  Free Adapter Blocking Reagent protocol to minimize presence of adaptor-dimers and index hopping rates.  The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2x150 cycles) of the Illumina NovaSeq6000. The instrument's computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated 2.5-3 billion paired-end reads (~950Gb) with an average Q30%>= 92.5% and Cluster PF= 85.4%.