Truseq stranded total rna sample kit

RNA sequencing was performed at the Johns Hopkins University School of Medicine Transcriptomics and Deep Sequencing Core, guided by the direction of Dr. Haiping Hao. RNA‐seq library for Illumina platform sequencing was prepared using Illumina TruSeq stranded total RNA Sample kit following manufacturer's recommended procedure. Briefly, 100 ng of total RNA was first depleted of ribosomal RNA with Ribozero Gold magnetic beads and further purified with Agencourt AMPure XP beads. RNA was fragmented at 94 °C for 8 min and primed with random primer. The fragmented RNA was then converted to double strand cDNA, end repaired, A tailed, and ligated with Unique Dual Indexed adaptors. The adaptor added cDNA library was then PCR amplified using the following conditions: 94 °C for 30 s, 15 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR amplified library was purified using Agencourt AMPure XP magnetic beads and run out on Agilent High Sensitivity DNA Chip for quality check. Individual library was then further quantified using KAPA library quantification qPCR kit and pooled. The pooled library was sequenced on Illumina NovaSeq S1 200cycle kit for 2X100bp sequencing.