title: GSM5542909: MA6-Hb45-BRU4: Pooled Male FHM High Brain RepB; Pimephales promelas; RNA-Seq
library_construction_protocol: Total RNA was extracted from tissue samples of individual fish using the RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany) in a QiaCube instrument (Qiagen).  Pooled samples with an RNA Integrity Number (RIN) ≥ 9 were used for mRNA sequencing Steps for generating libraries from total RNA included the following: mRNA enrichment was performed using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs, Ipswich, MA, USA); cDNA synthesis was done using NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules (New England BioLabs).  Subsequent steps of library preparation were done using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs).  Adapters and PCR primers were purchased from New England BioLabs.  Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems, Wilmington, MA, USA).  Determination of average fragment size was done with a LabChip GX (PerkinElmer, Waltham, MA, USA) instrument.   Once libraries were normalized and pooled, they denatured in 0.02N NaOH and neutralized using HT1 buffer.  The pool was loaded at 225pM on an Illumina NovaSeq (Illumina, San Diego, CA, USA) S4 lane using Xp protocol.  The run was performed for 2x100 cycles (paired-end mode).  A phiX library was used as a control and mixed with libraries at 1% level.  Base calling was performed with RTA v3.4.4. Program bcl2fastq2 v2.20 was applied to demultiplex samples and generate fastq reads.