GSM5070526: ChIPseq-Input-testis; Macaca mulatta; ChIP-Seq
In brief, testis frozen tissues were cut into small pieces on dry ice and collected to 1.7 ml tubes containing cold PBS in which samples were cross-linked with 2% (w/v) formaldehyde at room temperature for 30 min using end-over-end tumbler. We then crushed fixed tissues in the presence of ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) by 40 strokes with a Dounce homogenizer pestle “B” (Kimble-Chase, Vineland, USA). To shear the chromatin into 150 to 200 bp, lysed samples were sonicated using Covaris ultrasonicator (Covaris, E220). Sonicated lysate was then diluted 1:10 with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-1001.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) and we performed immunoprecipitation using 5 µg anti-TCFL5 antibody (Sigma, HPA055223, lot # 000000907). Following the immunoprecipitation of the chromatin, we extracted DNA by phenol:chloroform:isoamyl alcohol (25:24:1) (pH 8). Library construction for input DNA was performed after DNA extraction as previously described (Zhang, Z., Theurkauf, W.E., Weng, Z., and Zamore, P.D. (2012). Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence 3, 9.).