GSM5268622: Refed_Surface3; Astyanax mexicanus; RNA-Seq
Total RNA was extracted using Trizol reagent (Ambion). For ChIP-seq, livers from eight juvenile fish (4-month-old) were pooled together and snap frozen. Then the frozen tissues were grinded into powder, followed by 1% pfa (diluted from 16% pfa, Thermo Fisher, PI28906) fixation for 10 min at room temperature. The cross link was quenched with 0.125 M glycine. Chromatin shearing was performed by using a Bioruptor sonication system with following parameters: 30 s on and 30 s off per cycle, 10 cycles in total. DNA fragments were collected and purified with MAGnify™ Chromatin Immunoprecipitation System (ThermoFisher, 492024) according to the kit instruction. Libraries for RNA-seq were prepared according to manufacturer's instructions using the TruSeq Stranded mRNA Prep Kit (Illumina). The resulting libraries were purified using the Agencourt AMPure XP system (Beckman Coulter) then quantified using a Bioanalyzer (Agilent Technologies) and a Qubit fluorometer (Life Technologies). Libraries for ChIP-seq were prepared using the KAPA HTP Library Prep Kit (Roche, Cat No. KK8234) with 15 cycles of PCR and using 1:125 dilution of NEXTflex DNA barcodes (Perkin Elmer, Cat No. NOVA-514104). The resulting libraries were purified using the Agencourt AMPure XP system (Beckman Coulter) then quantified using a Bioanalyzer (Agilent Technologies) and a Qubit fluorometer (Life Technologies). Post amplification size selection was performed on all libraries using a PippinHT (Sage Science). Libraries for RNA-seq were re-quantified, normalized, pooled and sequenced on an Illumina HiSeq 2500 using v4 High Output chemistry, single read 50bp, RTA v1.18.64, and bcl2fastq2 v2.20 for demultiplexing and FASTQ file generation. Libraries for ChIP-seq were performed high throughput sequencing on the Illumina NextSeq platform.