title: GSM4459861: RNAseq.GPX4KO.mouse2.786O.left.rep2_FR2dL; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA samples were extracted using the Rneasy Mini Kit (Qiagen) following manufacturer's instructions. Genomic DNA samples were extracted using the QIAamp Blood Mini DNA isolation kit (Qiagen) from manufacturer's instructions. RNA sequencing library was prepared using NEB-Next Ultra RNA Library Prep Kit following the manufacturer's recommendations. Briefly, total RNA samples were quantified and tested for contamination and degradation. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo (dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. The qualified libraries are fed into Illumina Novaseq6000 sequencers after pooling according to its effective concentration and expected data volume using a 2x150 bp paired-end configuration.  Image analysis and base calling were conducted by the HiSeq Control Software. Raw sequence data generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.   To make Exome-sequencing library, DNA degradation and contamination were monitored on 1% agarose gels, and DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 1.0 μg genomic DNA per sample was used as input material for the DNA library preparation. Sequencing libraries were generated using Agilent SureSelect Human All Exon kit (Agilent Technologies, CA, USA) following manufacturer's recommendations and index codes were added to each sample. Briefly, fragmentation was carried out by a hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate 180-280bp fragments. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 3' ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated adapter molecules on both ends were selectively enriched in a PCR reaction. After PCR reaction, library hybridize with Liquid phase with biotin labeled probe, then use magnetic beads with streptomycin to capture the exons. Captured libraries were enriched in a PCR reaction to add index tags to prepare for hybridization. Products were purified using AMPure XP system (Beckman Coulter, Beverly, USA) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. Sequencing libraries that passed quality threshold were sequenced on an Illumina Novaseq6000 platform according to effective concentration and data volume.