Bioruptor sonication system ucd300

We used the Smart-seq2 protocol for full-length scRNA-seq according to the manufacturer’s instructions [61] (link). Briefly, single cell was transferred to lysis buffer with RNase inhibitor in a 0.2-ml PCR tube by mouth pipetting. First-strand cDNA synthesis was performed using a 25-bp oligo(dT)₃₀VN primer for 3′ amplification. PCR products were used to generate second-strand cDNA. After annealing to an index primer, the second-strand cDNA was fragmented into 350-bp pieces by a Bioruptor Sonication System (UCD300, Diagenode, Brussels, Belgium), and the reactions were purified by incubation with Ampure XP beads (Beckman, A63880, Fullerton, California, USA) at room temperature for 5 min. After quality inspection using an Agilent 2100 High Sensitivity DNA Assay Kit (Catolog No. 5067–4626, Agilent, Santa Rosa, CA) based on the manufacturer’s instructions, sequencing was performed on an Illumina HiSeq 2000 platform using 150-bp paired-end sequencing via the Smart-seq2 protocol.

A chromatin immunoprecipitation (ChIP) assay was performed as previously described (34 (link)). Briefly, lysates from stable nanobody-expressing U2OS cell lines were sonicated with a Bioruptor Sonication System UCD-300 (Diagenode, Seraign, Belgium) 21 times for 30 s with intermittent cooling. Anti-p53 (DO1) was used to perform the precipitation of the DNA-bound p53, whereas anti-NTF2 was used as an aspecific control. The chromatin-antibody protein complex was eluted from the protein-G-Sepharose beads with freshly prepared elution buffer (0.1 M NaHCO3 and 1% SDS). Reverse crosslinking and RNase treatment of the ChIP eluate were simultaneously performed overnight at 65°C. The samples were digested with proteinase K for 3 h at 50°C. The DNA was finally purified using QIAquick PCR Purification Kit (Qiagen, Venlo, The Netherlands). The eluted DNA was subsequently submitted to a RTqPCR (see above). The sequences of the primers used can be found in Supplementary Table S4 (35 (link)).