ChIP-Seq Input control: human glioblastoma from 64 year old male: Sample Gbm5_input
Tumor tissue preparation and chromatin immunoprecipitations were performed as described at https://doi.org/10.15781/T2251FR4Z. Samples were then brought to 25°C and residual RNA removed by adding 5 µl 0.5 mg/mL RNase A and incubating for 30 minutes at 37°C. Proteins were digested by adding 20 µl of 1 M Tris pH 6.8, 10 µl 0.5 M EDTA pH 8.0, and 3 µl 20 mg/mL Protease K, followed by incubation at 55°C for one hour. Samples were extracted with phenol-chloroform and precipitated with ethanol. The DNA pellet was resuspended in 15 µl sterile DNase and RNase free water (Ambion), and quantitated using a Qubit HS DNA kit (Q32851, Life Technologies). After assessing qPCR enrichment at positive control target sites, libraries were prepared using the NEBNext library prep kit (E6240L, New England Biolabs), with the following modifications: adapter ligation was performed before size selection; Bioo adapters (514103, Bioo Scientific), and Ampure XP beads (A63881, Beckman-Coulter) were used in place of columns for purification and size selection; PCR amplifications were minimized, often as low as 6-8 cycles when feasible.
UTEXAS-phs001389
Gbm5_input