GSM5628244: DJ27_Heart_RNA_AAV-Null_S38; Mus musculus; RNA-Seq
Tissues that were snap frozen in liquid nitrogen were removed from -80C. Tissues were homogenized in trizol with the bullet blender at 4C. RNA extraction was done with Qiagen RNeasy Kit. Samples were DNAse treated with the ThermoFisher Turbo DNA Free. The concentration and quality of the RNA of the samples was first assesed with the Agilent 2100 BioAnalyzer. An RNA Integrity Number (RIN) of 5 or higher was required to pass the quality control but a RIN of 9 or higher was desired. The 100ng of RNA was used for cDNA library preparation which included mRNA purification/enrichment, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and application following KAPA mRNA Hyperprep Kit prep (KK8581). Each resulting indexed library was quantified and its quality accessed by NovaSeq 6000; 5µL of 2nM pooled libraries per lane were denatured, neutralized, and applied to the cBot for flow cell deposition and cluster amplification before loading to HiSeq 3000/4000 for 75bp paired-end sequencing (Illumnia, Inc). Approximately 50M reads per library were generated. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy). The sequence reads were mapped to the designated reference genome using STAR (Spliced Transcripts Alignment to a Reference).