| 生长方案: |
After 24 hours of culturing, fibroblast cell were adhered to the culture plates and the RPMI medium was refreshed to wash away debris, dead cell and non-adherent cell. After cell reached a minimum of 80% confluency, they were washed with HBSS (Lonza BioWhittaker, Switzerland) and then detached from the culture plates through a ten-minute trypsin wash (10x diluted in HBSS) at 37°C. The fibroblasts were then first plated in T25 flasks after which samples in later passages were transferred to T75 flasks (VWR, Pennsylvania, USA; Tissue Culture Flasks 25 cm2 & 75 cm2) to allow for further expansion. During passages 1 through 5, cell have been harvested for downstream DNA and RNA analysis. For RNA isolation were stored in RNAlater (Thermo Fisher Scientific, |
| 处理方案: |
Mucosal samples were thoroughly washed in repeated cycles of ice-cold phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin and 40 μg/mlL G418 (PGA). The mucosa was then finely cut and placed in full-grown RMPI 1640 culture medium (Invitrogen) supplemented with 1.5 mg/mL collagenase A (Roche, Germany) and minced using the Gentlemacs Dissociator (Miltenyi Biotec, Leiden, the Netherlands). After 60 minutes of incubation at 37 °C, the Gentlemacs Dissociator was used a second time for further dissociation. cell were transferred to tubes and washed extensively with PGA after which the cell were plated and cultured at 37°C in RMPI 1640 with 10% FCS, 1% penicillin/streptomycin, 1% L-glutamin, G418 (40 μg/mlL; Lonza, Leusden, the Netherlands) and amphotericin B (0.025 μg/mL; Gibco, Rockford, IL). |
| 提取方案: |
RNA was isolated from the fibroblasts using the RNAeasy mini kit (Qiagen).The NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs) was used for mRNA isolation, cDNA generation and sequencing adapter ligation. Performed on an Illumina NextSeq500 at a coverage of 10M reads per sample. The preparation and sequencing of the RNA was performed at GenomeScan B.V. in Leiden, the Netherlands. |
| 建库方案: |
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