title: GSM3500809: GCM-treated_rat_microglia_DNaseI-seq_rep2; Rattus norvegicus; DNase-Hypersensitivity
library_construction_protocol: Briefly, 10^6 – 5 x 10^6 microglial cells (the same number of cells for each condition) were stimulated for 6 h with 100 ng/ml LPS, GCM or left untreated. Then, cells were lysed, nuclei were collected  and digested 10 min at room temperature with a series of DNaseI dilutions (0.2-3.2 U/reaction, Roche Diagnostics). Digested DNA was enclosed in low melting point (LMP) agarose blocks. DNAse-I activity at different concentrations was determined using pulsed field gel electrophoresis (PFGE) and the CHEF-DRII apparatus (Biorad),  and DNAseI activity at three optimal concentrations was selected  for further processing. DNA fragment ends were repaired and a “linker 1” (HPLC-purified,  annealed oligonucleotides: AGTTCCTTGGCACCCGAGAATTCCATCCGAC and GTCGGATGGAATTCTCGGGTGCCAAGG) using T4 DNA ligase (1 U/μL) and ligation buffer (10X) (Roche Diagnostics).  DNA with a ligated “linker 1” was isolated from agarose blocks and digested with MmeI enzyme (2 U/μL; New England Biolabs). Fragments containing “linker1” were purified using streptavidin beads (Dynal M-280; Invitrogen). Subsequently, a “linker 2” (HPLC-purified,  annealed oligonucleotides: GATCGTCGGACTGTAGAACTCTGAAC and GTTCAGAGTTCTACAGTCCGACGATCNN) was ligated to DNA on beads and the beads were treated with 0.15 M NaOH to denature DNA. The obtained DNA fragments attached to the streptavidin beads were used as a template for PCR reaction performed with primers containing the standard Ilumina library preparation sequences and indexes. PCR products were resolved in 3% agarose gels and purified from gel using DEAE-cellulose chromatography papers, and sequenced on Illumina Hi-seq 1500.
attributes.tag: GEO Accession
attributes.value: GSM3500809