title: GSM5111051: miaMBF rep mia3; Danio rerio; RNA-Seq
library_construction_protocol: 5dpf zebrafish larvae ( wild typeor mia40-/-) were anaesthetized using Tricaine, washed with Ringer's solution and homogenized. The homogenate (Whole) was centrifuged twice at 1500 Xg for 10 min to remove nuclei and debris which resulted in obtaining the post-nuclear supernatant (PNS). The PNS was centrifuged at 14,000 Xg for 15 min to pellet down Membrane Bound Fraction (MBF). Subsequently, the ultra-centrifugation of the supernatant at 100,000 X g for 60 min was performed to obtain the High-Speed Fraction (HSF).  RNA was extracted from Whole, MBF and HSF using the TRI Reagent solutionand isolated using isopropanol precipitation. The samples were treated with RNase-Free DNase (Qiagen, 79254) to avoid gDNA contamination and cleaned and concentrated using Agencourt RNAClean XP beads (Beckman Coulter, A63987). The RNA concentration was estimated by Quantus Fluorometer (Promega) or Nanodrop Spectrophotometer (ThermoFisher Scientific) and the integrity of RNA was verified by 2200 Tapestation system (Agilent Technologies). The RNA samples were stored in -80º C until further use. 2 μg ofRNA from each sample was spiked in with ERCC ExFold RNA Spike-In mixes (Thermofisher Scientific,4456739) following the manufacturer's guideline and subjected to mRNA enrichment by Poly (A) capture coupled with library preparation by using KAPA Stranded mRNA -Seq Kit with KAPA mRNA capture Kit (Kapa biosystems, KK8420) and following manufacturer's protocol.  Library fragment size distribution was confirmed by electrophoresis in Tapestation (Agilent Technologies). Library concentration was determined by both Quantus and qPCR. Sequencing was performed on the NextSeq500 instrument (Illumina) using the v2 chemistry, resulting in an average of 100 M of 2 x 75 bp paired-end reads per library.