title: GSM1700559: GR_032015; Homo sapiens; OTHER
library_construction_protocol: Library design: An initial experiment (Dec, 2014) was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 (run in March 2015) sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Library construction: ssDNA oligo containing the DNA binding site of interest was ordered from IDT, with flanking Illumina PE1 and PE2 sequences on each side for final PCR enrichment. FAM-labelled reversed PE2 primer was used to anneal/extend the ssDNA template to make dsDNA pools. Afterwards ExoI nuclease treatment followed by Qiagen PCR column purification can make sure only dsDNA templates exist in the pool. DNA separation, extraction, and sample preparation protocol: Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Gel bands containing each DNA fragment were cut, extracted by acrylamide extraction buffer. No more than 10 rounds of PCR by Illumina PE1-Genetics, indexed PE2 primers can enrich each portion of DNA fragments. All indexed (barcoded) DNA samples were pooled together for Illumina sequencing.
identifiers.namespace: GEO
identifiers.id: GSM1700559