GSM9083100: C301coated_silicone_3; Homo sapiens; RNA-Seq
Cell culture medium was removed from the wells 24 hours after PMA removal and the cells were disrupted by adding buffer RLT (79216, Qiagen), containing 1% 2-mercaptoethanol (63689, Sigma-Aldrich), onto the cells (1200 μL for breast implant surfaces in 12-well plates, 300 μL for PAA gels and surgical meshes, 350 µL for other materials in 24-well plates). Cells were resuspended by vigorous pipetting, vortexing for 1 minute and transferred to polypropylene vials (DNA LoBind tubes, 0030108051, Eppendorf). Vials were immediately stored at -80 °C until further processing. For the surgical meshes, 300 μL of lysate from 2 wells was pooled into one vial, creating a total of 3 samples from 6 wells. For the PAA gels, 300 µL buffer was added for 10 minutes on ice, followed by vigorous pipetting and transferral to vials. Subsequently, this procedure was repeated on the same well with another 300 µl of RLT buffer to retrieve RNA absorbed into the gel. Total RNA extraction was performed using the RNeasy Mini Kit (74104, Qiagen), in combination with a QIAshredder homogenisation (79656, Qiagen) and DNase digestion step (79254, Qiagen), following the manufacturer's protocol. The RNA concentration was determined using the Qubit RNA HS assay (Q32852, Invitrogen) on a Qubit 3 fluorometer (Invitrogen). RNA quality was analysed using the 2100 Bioanalyzer (Agilent Technologies) and the RNA 6000 Nano kit (5067-1511, Agilent Technologies). All RNA samples had an RNA Integrity Number (RIN) of 9.5 or higher and were considered of sufficient quality for sequencing. Three samples of each experimental group were further processed for Next Generation Sequencing using the TruSeq Stranded mRNA kit (20020594, Illumina), following the Low Sample Protocol. Briefly, samples were enriched for poly-A RNA, after which first-strand cDNA synthesis was performed using SuperScript™ II Reverse Transcriptase (18064014, Thermo Fisher Scientific). TruSeq RNA Single Indexes Set A and B (20020492 and 20020493, Illumina) were used for sample indexing. AMPure XP beads (A63881, Beckman Coulter) were used for all purification steps during the library preparation. Size and purity of the libraries were assessed using a 2100 Bioanalyzer with the DNA 1000 Kit (5067-1504, Agilent Technologies). Sequencing libraries were normalised based on KAPA qPCR quantification (7960140001, Roche Diagnostics) using a QuantStudio 6 Flex Real-Time PCR System (ThermoFisher Scientific) to ensure equal amounts of DNA before pooling. Sequencing was performed on a NextSeq 550 System (Illumina) using the NextSeq 500/550 High Output Kit v2.5 (75 cycles) in three runs, with 16 libraries per run.