GSM5597345: P. gingivalis persisters-treated HGECs - Tb_3; Homo sapiens; RNA-Seq
Total RNA was purified with RNeasy Plus Mini Kit (Qiagen, Benelux BV Qiagen GmbH, Hilden, Germany) according to the standard procedures. RNA libraries were prepared for sequencing using standard Illumina protocols. Briefly, NanoPhotometer® spectrometer (IMPLEN, CA, USA) was used to detect the quantity and quality of the RNA. RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the RNA fragment size. Poly-T oligo-attached magnetic beads were used to purify mRNA from total RNA. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) and index codes were added to attribute sequences to each sample. Library RNA concentration was measured using Qubit® RNA Assay Kit (Life technologies, Waltham, MA, USA) in Qubit® 3.0 to preliminary quantify and then dilute to 1ng/μl. Insert size was assessed using the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA), and qualified insert size was accurately quantified using CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) to make sure it match the library valid concentration (＞10 nM). The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina, Santiago, CA, USA). After cluster generation, the libraries were sequenced on a NovaSeq 6000 platform (Illumina, Santiago, CA, USA), and 150 bp paired-end reads were generated.