ATAC-seq of primary human fibroblasts
ATAC-seq of primary human fibroblasts
Cell monolayers were trypsinized, neutralized with medium, washed with PBS, and centrifuged. Washed cell pellets were resuspended in Macherey-Nagel Nucleospin RNA Plus Lysis Buffer. No dissections were performed so this protocol is not applicable. Cells were grown in DMEM + 10% fetal bovine serum + sodium pyruvate + Gluta-Max + penicillin/streptomycin in a 5% 02 + 5% CO2 incubator at 37°C. For X-ray induced senescence, confluent cells in a 10 cm plate were irradiated with 40 Gy of 120 kV X-rays 20 or 40 Gy X-rays at 120 kV generated by a Faxitron 43855-D irradiator. The next day, cells were trypsinized and split into 3 x 10 cm dishes and incubated for 10 days before performing ATAC-seq. For some experiments, cells were irradiated with 40 Gy X-rays as above and incubated for 9 days. They were then treated with 20 pg/ml of recombinant IL1-alpha or IL1_beta for 24 hours before performing ATAC-seq. Finally, for some experiments, proliferating sub_confluent fibroblasts were treated with 20 pg/ml of recombinant IL1_alpha for 24 hours before performing ATAC_seq. The following software were used for data conversion: Bowtie2, sambamba, samtools, and deepTools. After the ATAC-seq tagmentation, the reaction was stopped by the addition of SDS to 0.1% final concentration and the addition of 10 midrograms of Proteinase K. The solution was incubated at 55°C for 15 mins, and DNA was then purified unsing the Macherey-Nagel PCR clean-up kit. The purified DNA was then amplified using Nextera-type PCR primers to add Illumina indexes for high_throughput sequencing. ATAC-seq was performed with the Omni-ATAC protocol as described in Nature Protocols 2022 (DOI: 10.1038/s41596-022-00692-9). Assembled Tn5 transposase was prepared as described (DOI: 10.1101/gr.177881.114). For some experiments, assembled Tn5 was bought from Active Motif or Diagenode.