title: GSM8689821: Pachon_old_starved_1; Astyanax mexicanus; RNA-Seq
library_construction_protocol: Total RNA was extracted using standard phenol/chloroform extraction. 1 ml of Trizol (15596026, Thermo Fisher Scientific) was used per 50 to 100 mg tissue. Samples were homogenized with Trizol and 3.0 mm TriplePure M-Bio Grade beads (D1132-30, Benchmark Scientific) on Beadbug 6 homogenizer (D1036, Benchmark Scientific). After a brief centrifugation, 200 µl of chloroform was added per 1 ml of Trizol, then vortex for 15 seconds and incubated at room temperature for 2 min. Samples were centrifuged at 12,000 g for 15 min at 4°C. Upper transparent aqueous phase was transferred carefully without disturbing the interphase into fresh tube and added with 500 µl of Isopropyl alcohol per 1 ml of Trizol used initially. 2 µl of GlycoBlue Coprecipitant (AM9515, Thermo Fisher Scientific) was added to visualize RNA pellets. Samples were incubated at room temperature for 10 min and centrifuged at 12,000 g for 10 min at 4°C. Supernatant was removed and RNA pellets were washed twice with 1 ml of 75% ethanol per 1 ml Trizol used initially. Samples were mixed by vortexing and centrifuged at 7400 g for 5 min at 4°C. RNA pellets were air dried for 15 min and dissolved in RNase free water. RNA solutions then went through on column purification and DNA digestion using RNeasy Mini Kit (74104, QIAGEN INC) and DNase kit (79254, QIAGEN INC). Total RNA-Seq libraries were generated from 3.8-500 ng of high-quality total RNA, as assessed using the Bioanalyzer (Agilent), according to the manufacturer's directions using a 200-5-fold dilution of the universal adaptor and 16-9 cycles of PCR per the respective input mass with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L, NEB), the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (E7400L, NEB), and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (E6440S, NEB) and purified using the SPRIselect bead-based reagent (B23318, Beckman Coulter). Resulting short fragment libraries were checked for quality and quantity using the Bioanalyzer and Qubit Fluorometer (Life Technologies).  Equal molar libraries were pooled, quantified, and converted to process on the Singular Genomics G4 with the SG Library Compatibility Kit, following the “Adapting Libraries for the G4 – Retaining Original Indices” protocol.  The converted pools were sequenced to the required number of reads per library on F2 flow cells (700102) or F3 flow cells (700126) on the G4 instrument with the PP1 and PP2 custom index primers included in the SG Library Compatibility Kit (700141), using Instrument Control Software 23.08.1-1 with the following read lengths: 8 bp Index1, 100 bp Read1, 8 bp Index2, and 100 bp Read2. Following sequencing, sgdemux 1.2.0 was run to demultiplex reads for all libraries and generate FASTQ files.