GSM5351581: run_06_05_17__R1_CEBPg_homo_75nM_lowBand__5mCG; Homo sapiens; OTHER
SELEX-seq: The Hth-Exd-Ubx SELEX experiment was carried out following previously published methods (Slattery, Cell, 2011). Briefly, after expressing and purifying the wild-type homeodomain proteins, a final concentration of ~50 nM was assembled, incubated with excess DNA (10-20 fold) for 30 minutes, and loaded onto an EMSA gel. A DNA library with 30 randomized bases was used. The TF-bound fraction was isolated from the gel, amplified, and either subjected to another round of enrichment or prepared for sequencing. An all, three rounds of enrichment were performed. After each selection round, the DNA was extracted from the gel and amplified by using Ilumina's small RNA primer sets. Sequencing barcodes were added in a five cycle PCR step and the final library was gel-purified using a native TBE-gel before sequencing. Libraries were sequenced at the New York Genome Center using separate lanes on an Illumina HiSeq 2000 sequencing machine. EpiSELEX-seq:  ATF4 protein purification and EpiSELEX-seq experiments were performed as described previously (Kribelbauer, Cell reports, 2017). Purified CEBPg protein was kindly donated by the Lomvardas lab at the Zuckerman Institute at Columbia University. To generate randomized 5hmC or 6mA libraries, single-stranded oligos with a 16-bp randomized region were ordered from TriLink Biotechnologies, substituting i) deoxycytidine triphosphate (dCTP) with deoxy-(5hm)-cytidine triphosphate (d5hmCTP), or ii) deoxyadenosine triphosphate (dATP) with deoxy-(6m)-adenosine triphosphate (d6ATP) during the synthesis step. For double-stranding, a standard mix of deoxy-nucleotides was used, resulting in hemi-modified libraries. 5mCpG libraries were generated by enzymatic treatment with M.SssI (NEB). The library sequences consisted of constant  left (GGTAGTGGAGG-) and right (-CCAGGGAGGTGGAGTAGG) adapters flanking a library specific barcode and a 16bp randomized sequence: -TGGG-CCTGG-N16- (no modification), -GCAC-CCTGG-N16- (5mCpG), -CAGT-CCTGG-N16- (5hmC), and -AGTG-CCTGG-N16- (6mA). K_D-seq: 6xHis tagged Drosophila Distalless (Dll) protein lacking amino acids N terminal to its homeodomain was purified by standard procedures. The 10mer R0 library was generated by annealing the library oligo GTTCAGAGTTCTACAGTCCGACCTGG-10N-CCAGGACTCGGACCTGGACTAGG and the SELEX-R primer CCTAGTCCAGGTCCGAGT, followed by a Klenow mediated primer extension reaction. The SELEX procedure was largely the same as previously described (Slattery, Cell, 2011), except that a Cy5 labeled DNA probe, instead of a P32 labeled probe, was used as the marker to indicate where the bound and unbound fractions were. For each SELEX condition, 15ul of protein solution (at 2x final concentration) in dialysis buffer (20mM HEPES pH8.0, 200mM NaCl, 10% glycerol, 2mM MgCl2, 0.05% Tween-20) was made. The library mixture was made by adding desired amount of the R0 library to 6ul of 5x binding buffer (50mM Tris-HCl pH7.5, 250mM NaCl, 5mM MgCl2, 20% glycerol, 2.5mM DTT, 2.5mM EDTA, ~125ng/ul polydIdC, 100ng/ul BSA, 0.125% Tween-20), and filling to 15ul with H2O. The protein and DNA parts were mixed and incubated at room temperature for 30 to 40 minutes before loading the gel. After running the gel, gel slices corresponding to the bound and unbound fractions were cut from the gel and purified. Each purified SELEX DNA was properly diluted such that the PCR program gave good library yield for all samples. The libraries were pooled and sequenced using Illumina Nextseq 550, following standard procedures. Kinase sequencing: The degenerate peptide library contained 11-residue sequences with five randomized amino acids flanking either side of a fixed central tyrosine residue: GCTGGCCAGTCTGGCCAG-NNSNNSNNSNNSNNStatNNSNNSNNSNNSNNS-GGAGGGCAGTCTGGGCAGTCTG. These sequences were fused to the eCPX bacterial surface display scaffold (Rice, Protein Engineering, Design & Selection, 2008). This construct was inserted into the SfiI-digested pBAD33-eCPX plasmid, as described previously (Shah, eLife, 2018). Transformed DH5a cells were grown overnight in liquid culture and the plasmid DNA library was extracted and purified using a commercial midiprep kit. A phosphotyrosine monoclonal antibody, pY20, conjugated to the fluorophore, perCP-eFluor 710 (Invitrogen), was biotinylated before use in the specificity screen. The catalytic domain of the human tyrosine kinase c-Src was screened against the degenerate peptide library as described previously (Shah, eLife, 2018), with one main difference being the use of magnetic beads to isolate phosphorylated cells, rather than fluorescence-activated cell sorting. Time points were taken at 5, 20, and 60 minutes. The phosphorylated cells were isolated with Dynabeads FlowComp Flexi (Invitrogen) following the manufacturer's protocol. In total, two populations were collected for each time point: cells that did not bind to the magnetic beads and eluted after each wash (unbound) and cells that bound to the magnetic beads and eluted after the addition of the release buffer (bound). After isolation of these two populations, the cell pellet was collected, lysed, and used as a template in a PCR reaction (Shah, eLife, 2018). The product of this PCR reaction was then used as a template for a second PCR reaction to append a unique 5' and 3' indices. Each sample was pooled to equal molarity and sequenced by paired-end Illumina sequencing on a MiSeq instrument.