Hitrap q hp anion exchange column

For small RNA sequencing, two replicates per strain were prepared. Small RNA was isolated from 50 pairs of ovaries using HiTrap Q HP anion exchange columns (Cytiva, Velizy-Villacoublay, France) as described in [37 (link)], and the eluate was run on a 10% TBE urea gel (Thermo Fisher Scientific). Small RNA size selection (18–50 bp) was performed on gel at the sequencing facility. Quality was checked with the Bioanalyzer small RNA kit (Agilent, Santa Clara, CA, USA). Library construction was performed using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA) and sequenced (1 × 50 single reads) on an Illumina HiSeq 4000 at the IGBMC Microarray and Sequencing facility. Adapter sequences were removed using cutadatp [38 (link)]. Size selection was then performed using PRINSEQ lite version 0.20.4 [39 (link)]. All subsequent analyses were built upon small RNA counts after normalization according to the miRNA amounts, as described in [34 (link)].

Bactotryptone and yeast extract were obtained from BD Biosciences (San Jose, CA, USA). Ni-nitrilotriacetic acid (NTA) agarose was obtained from Qiagen (Hilden, Germany), and frTet (4-(1,2,3,4-tetrazin-3-yl) phenylalanine) was purchased from Aldlab Chemicals (Woburn, MA, USA). TCO–Cy3 was purchased from AAT Bioquest (Sunnyvale, CA, USA). Axially substituted trans-cyclooctene maleimide (TCO-maleimide, A) was purchased from FutureChem (Seoul, Korea). Disposable PD-10 desalting columns, HiTrap Q HP anion exchange columns, and Superdex 200 10/300 GL Increase size exclusion columns were purchased from Cytiva (Uppsala, Sweden). All other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated.

The human p300(BRPH_ΔAILZ) (aa 1035–1720) core was cloned into the pGEX-6P-1 vector with an N-terminal GST tag and a Pre-Scission cleavage site. The flexible loop of residues 1520 to 1581 was replaced by an SGGSG linker, and the single mutation Y1467F was also introduced to stabilize the p300 core, based on former studies (Zhang et al., 2018 (link)). The histidine tag was added to the C-terminus of the core domain to increase the yield. The proteins were expressed in E. coli BL21-CodonPlus (DE3)-RIL competent cells in LB medium supplemented with 0.05 mM ZnCl₂. Protein expression was induced with 0.5 mM IPTG for 20 h at 16°C. The proteins were purified with glutathione agarose in 50 mM Tris-HCl (pH 7.5) buffer, supplemented with 500 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, and 5 mM β-mercaptoethanol. The GST tag was removed by overnight digestion at 4°C with Pre-Scission protease. The proteins were further purified by ion exchange on a HiTrap Q HP anion exchange column (Cytiva) and by size exclusion chromatography with a HiLoad 16/600 Superdex 75 column (Cytiva), in buffer [20 mM HEPES (pH 7.5) 200 mM NaCl, and 2 mM dithiothreitol]. All mutants were generated using either a QuikChange Site-Directed Mutagenesis Kit (Agilent) or a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturers’ protocols, and then grown and purified as described above.