| 生长方案: |
PBMCs were isolated by differential centrifugation over Ficoll-Paque (Lymphoprep, Stemcell Technologies). Cells were washed three times in PBS. PBMCs and monocytes were resuspended in RPMI culture medium supplemented with 2mM glutamax, 1mM pyruvate and penicillin/streptomycin (all from Thermo Fisher Scientific) and counted on a Casy counter. Monocytes were isolated using negative MACS isolation with the Pan monocyte isolation kit (Miltenyi Biotech). Briefly, stimulated PBMCs were washed with PBS and incubated with versene solution (0.48mM EDTA, Sigma Aldrich) for 30 minutes at 37 . Cells were scraped from the plates, counted, spun down and resuspended in MACS isolation buffer (PBS with 0.5% BSA and 2mM EDTA). Monocytes from COVID-19 patients were isolated directly after isolation of PBMCs. PBMCs were counted, spun down and resuspended in MACS isolation buffer. Monocyte isolation was performed according to manufacturer instructions |
| 处理方案: |
Human PBMCs were trained as described before. In short, 500.000 PBMCs were added into 96-well flat bottom plates. Cells were allowed to adhere for 1h at 37. Cells were washed three times with PBS prior to stimulations. After washing cells were incubated with culture medium only as negative control, or treated with 100 M chloroquine (Sigma Aldrich), 100 hydroxychloroquine (Sigma Aldrich) or 0.01 rapamycin (Selckchem) for 1 hour at 37. Subsequently cells were incubated with 105 cells/ml HKCA (Invivogen) for 24 hours together with the respective treatment for 24 hours at 37 Subsequently, cells were washed and cells were rested for five days in RPMI culture medium containing 10% FBS. After the resting period cells were stimulated with either RPMI as negative control, 10ng/ml LPS (Sigma Aldrich) or 1ug/ml Pam3CSK4 (Invivogen). |
| 提取方案: |
For RNA isolation 1*106 isolated monocytes were resuspended in 350 of RNA later Buffer (Qiagen). RNA was isolated using RNeasy kit (Qiagen) including DNAseI (Qiagen) digestions. For ChIP-seq, isolated monocytes were resuspended in RPMI culture medium and fixed using formaldehyde (1% final concentration, Sigma Aldrich) for 10 minutes at room temperature. Unreacted formaldehyde was quenched with 125 mM glycine and incubated for 5 minutes at room temperature. Cells were washed twice in PBS containing protease inhibitor cocktail (Roche) and 1 mM PMSF (Roche), and subsequently snap frozen in liquid nitrogen. Cell pellets were stored at -80 for further use. Cells were sonicated at a concentration of 15 million cells/ml using a Bioruptor pico sonicator (Diagenode; 10 cycles, 30s on, 30s off, at 4 . Immunoprecipitation was performed using the MagnaChIP kit (Merck Millipore) according to manufacturer instruction. In short, 500,000 cells were incubated overnight with 1 H3K4me3 or H3K27Ac antibody (Diagenode) and protein A magnetic beads at 4. Beads and chromatin/antibody mixture were washed four times for 5 minutes at 4. After washing chromatin was eluted and proteins were degraded using proteinase K. DNA was purified using spin columns and eluted in millliQ. |
| 建库方案: |
Total RNA isolated from monocytes was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems). In short, oligo hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94 C for 6 min. First strand synthesis, second strand synthesis and A-tailing was performed according to protocol. For the adapter ligation, a 1.5 M stock was used (NextFlex DNA barcodes, Bioo Scientific). First and second post-ligation cleanup was performed according protocol. A total of 11 PCR cycles were performed for library amplification. The library amplification cleanup was done using a 0.8x followed by a 1.0x beadbased cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer kit, and the library concentration was measured using the dsDNA High Sensitivity Assay 17 (Denovix). Paired-end sequencing reads of 50 bp were generated using an Illumina NextSeq 500. ChIP-seq libraries were prepared using the Kapa Hyper Prep Kit according to manufacturer protocol, with the following modifications. 2.5 L of the NEXTflex adapter stock (600 nM, Bioo Scientific) was used for adapter ligation of each sample. Libraries were amplified with 12-15 PCR cycles followed by a double post-amplification clean-up was used to ensure proper removal of adapters. Samples were analyzed for purity using a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent). Libraries were paired-end sequenced to a read length of 50 bp on an Illumina NextSeq500. |
