GSM8585788: skin_BT.group_s7_1_BT9; Homo sapiens; RNA-Seq
Frozen leprosy skin biopsy specimens were cut into forty 4µm sections and lysed in RLT Buffer (Qiagen, cat n°79216) supplemented with 1% β-mercaptoethanol, followed by immediate storage at -80°C. For RNA extration, samples were added to Lysing Matrix B tubes containing 100µm zirconium beads (OPS Diagnostics, cat n°PFLB 100-100 06U) and sonicated in FastPrep®-24 instrument (MP Biomedicals) at 4°C for 2 cycles of 45 seconds at 4m/s with five minutes intervals on ice after each cycle. Total RNA was then isolated using the RNeasy Micro kit (Qiagen, cat n°74004) according to the manufacturer's protocol and quantified by NanoDrop spectrophotomer and Qubit Fluorometer. All samples had RNA integrity numbers above 8.0 as determined by Bioanalyzer quality assessment (Agilent Technologies). Depletion of ribosomal RNA and library preparation were performed using Ribozero Gold Human/Rat/Mouse (Illumina, cat n°MRZG126) and KAPA Stranded RNA-Seq Library prep Kit (Kapa Biosystems, cat n°KR0934) according to the manufacturer's protocols. Final libraries were reassessed for quality (Qubit and Bioanalyzer), barcoded, multiplexed at eight samples per lane (10µM each library), and sequenced on a HiSeq 4000 sequencer (Illumina) at single-end 100bp reads.