Truseq stranded total rna library prep kit with ribo zero

RNA library preparation and sequencing were completed at Expression Analysis (10/2018, EA Genomic Services, Q2 Solutions—a Quintiles Quest Joint Venture, Durham, NC), as described previously^{14 (link)}. FFPE RNA underwent reduced (or no) fragmentation during library preparation, depending on Agilent Bioanalyzer profiles. RNA was ribo-depleted and cDNA libraries were synthesized using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (catalog no. RS-122-2303; Illumina, San Diego, CA). Paired-end 50 base pair sequencing to at least 25 million reads per sample was performed on Illumina HiSeq 2500 instruments. Mean sequencing depth was 29.2 to 30.0 million reads within each tissue and preservation group with an average read Phred score of 35.9 to 36.2 per tissue and preservation group (Supplementary Table 9). Base call files were transformed into FASTQ files via bcl2fastq version 2.20 (Illumina). See Supplementary Table 9 for RNA pre-alignment quality metrics by individual sample.

RNA was extracted using a Trizol/RNeasy hybrid protocol. In brief, after phase separation, the aqueous phase was mixed with one volume of 70% ethanol and passed through an RNeasy spin column (QIAGEN RNeasy Mini Kit). RNA quality was determined using the high-sensitivity RNA ScreenTape assay (Agilent 5067-5579), and all samples had RIN scores >7.0. RNA-seq libraries for differential expression or splicing analysis were prepared for four biological replicates/conditions, including four young adult males, four aged males, four young adult females, and four aged females per library type. For differential expression analysis, total RNA libraries were prepared using the TruSeq Stranded Total RNA library prep kit with Ribo-Zero (Illumina 20020596). Paired-end 100 bp sequencing to a depth of ~50 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Broad Stem Cell Research Center Sequencing Core. For splicing analysis, mRNA libraries were prepared with poly-A selection using the TruSeq Stranded mRNA library prep kit (Illumina 20020594), and paired-end 75 bp sequencing to a depth of ~50 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Neuroscience Genomics Core.

Ten to 20 manually dechorionated 24 hpf embryos of a germline mutant incross and a germline wild-type incross were homogenized in TRIzol (Ambion, 15596018). For 0 and 3.3 hpf, 20 non-dechorionated embryos were collected and homogenized in TRIzol. Subsequently, the Quick RNA microprep kit (Zymo Research, R1051) was used to isolate RNA and treat the samples with DNAseI. Samples were depleted from rRNA using the Ribo-Zero rRNA Removal Kit (Illumina, MRZH11124), followed by fragmentation and cDNA synthesis, and libraries were generated using the KAPA Hyper Prep Kit (KAPABiosystems, KK8504). Sequencing libraries were paired-end sequenced (43 bp read-length) on an Illumina NextSeq500 platform. However, two samples per genotype at 24 hpf were generated with the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina, RS-122-2201) and single-end sequenced (50 bp read-length) on an Illumina HiSeq 2500. For wild-type and MZezh2 mutant embryos, six and seven biological replicates were used, respectively.