Rprotein a sepharose bead

ChIP for FS2-tagged AEBP2 was performed as described (Farcas et al., 2012 (link)) except that we used rProtein A Sepharose beads (GE Healthcare) that had been blocked for 1 h at 4°C with 1 mg/ml bovine serum albumin and 1 mg/ml yeast tRNA (Sigma). ChIP for endogenous proteins was performed as described (Stock et al., 2007 (link)), except approximate concentration after sonication was measured by measuring absorbance using the Nanodrop ND-1000 (ThermoScientific) and 200 µg of chromatin was used per immunoprecipitation and after the washes DNA was purified using ChIP DNA Clean & Concentration columns (Zymo Research) and eluted in 10 µl volume. Preparation of ChIP-seq libraries and bioinformatics analysis is described in the supplementary Materials and Methods.

Tuber samples, which were used for starch and sugar contents analysis, were used for both RNA-seq and ChIP-seq. Tubers harvested from six individual plants were pooled as a biological replicate for each line. Two biological replicates of RNA-seq libraries were constructed for each temperature treatment and sequenced using an Illumina NovaSeq 6000 platform with 150 bp PE mode. All Illumina sequencing in this study was conducted using the same platform and mode in Novagene.
ChIP experiments were conducted following the published protocol [75 (link)], with antibodies against H3K4me3 (Abcam 8580), H3K27me3 (Millipore 07–449) and H3K27ac (Millipore 07–360), respectively. To achieve the highest resolution, chromatin was digested into monomer nucleosome pattern (~150 bp fragments) using MNase (Sigma N3755). Antibody-captured chromatin complex was precipitated using rProtein A sepharose beads (GE 17–1279-01). ChIP-DNA was separated from the precipitated chromatin for ChIP-seq library construction and sequencing.

Both leaves and roots were used for RNA-seq and ChIP-seq, respectively. Each tissue from three individual plants were pooled together as a biological replicate for each line. Two biological replicates of RNA-seq libraries were developed and sequenced using an Illumina NovaSeq 6000 platform with the mode of 150 pair end (PE) sequencing. All RNA-seq libraries were developed and sequenced in Novagene company.
According to the published protocol (Zhang et al., 2012 (link)), the ChIP experiments were performed with the antibody against H3K27me3 (Millipore 07–449). Chromatin was digested into monomer nucleosome pattern (∼150 bp fragments) using MNase (Sigma N3755) to obtain the highest resolution of the histone modification signal. Chromatin in monomer nucleosome pattern carrying H3K27me3 were captured and precipitated using rProtein A Sepharose beads (GE 17-1279-01), followed by ChIP-DNA separation. The isolated ChIP-DNA was applied for library construction, which was subsequently sequenced using the same method as the RNA-seq libraries in Novagene company.

BALB/c mice 5 to 6 week old were used for immunization. Groups (n=5 per group) of mice were immunized subcutaneously with 50 μg of Hsc70-P19, Hsc70-P26, or Hsc70-control fusion protein emulsified in complete Freund's adjuvant at the first time. Mice were boosted 3 times with the fusion protein emulsified in incomplete Freund's adjuvant at 3-week intervals [29 (link), 30 (link)]. Blood were taken from the tail vein on day 0 and on day 7 after every immunization. Serum was pooled from each group of mice by mixing equal volumes of fourth immune serum of each mouse. And then the sera were used for IgG purification by using rProtein A sepharose beads (GE Healthcare). The purified IgG were used for further ADCC, CDC, and cell proliferation assays.
To test the antibody responses against peptides and EGFR in the immunized mice, ELISA was performed as previously described [26 (link)]. Briefly, 96-well plates were coated with GST fusion protein or rhEGFR, and then the dilutions of serum from immunized mouse were added. The plate coated with GST was used as negative control. Bound antibodies were detected with HRP-conjugated goat anti-mouse IgG (Beijing Zhongshan Golden Bridge Biotechnology Co Ltd, China). The reaction was developed with o-phenylenediamine as substrate. OD₄₉₀ was measured by using a microplate reader.