title: GSM3141939: input DNA_Rep1; Mus musculus; ChIP-Seq
library_construction_protocol: E17.5 stomachs were dissected, cut into small pieces, and crosslinked in solution A (1% formaldehyde, 50mM Hepes-KOH, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA) for 20 minutes at room temperature. 1/20 volume of 2.5 M glycine was added to quench formaldehyde. Tissues were rinsed in ice-cold PBS and dounced in Wheaton tissue grinder. Cell suspensions were passed through a 100μm cell strainer to remove connective tissues, then centrifuged at 4 °C at 2500 × rcf for 3 min, followed by a wash in PBS. Gli2, H27K27ac, or H3K36me3 antibodies were incubated with pre-blocked magnetic beads in 250μL total volume for 8 hours at 4 °C, then washed and resuspended in 100μL 0.5% BSA (w/v) solution. Cell pellets were lysed by sequentially resuspending and centrifuging in LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100), and LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA). Pellet was resuspended in LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine) and sonicated into 200-400bp fragments. Sonicated lysates were diluted with LB3. After saving 50μL for input control, they were split into tubes containing 10% v/v Triton X-100 with Gli2 (homemade), H3K27ac (Millipore 05-1334 lot 2489078), or H3K36me3 (Abcam ab9050 lot GR204353-1) antibody, and incubated overnight. Beads were washed 6 times in RIPA buffer (50 mM Hepes-KOH, pH 7.5; 500 mM LiCl; 1mM EDTA; 1% NP-40 or Igepal CA-630; 0.7% Na-Deoxycholate), and once in TBS (20 mM Tris-HCl, pH 7.6; 150 mM NaCl). Protein-DNA complexes were eluted and crosslink reversed with 200 μL of elution buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA; 1% SDS). The 50μL input lysate was also included in this step. Eluted DNA was diluted with 200 μL of TE, and treated with RNaseA for 30 min at 37°C, and subsequently with proteinase K for 2 hours at 55°C. Phenol:chloroform:isoamyl alcohol was used to extract DNA. Libraries were prepared according to manfacturer's instructions for the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit.
attributes.tag: GEO Accession
attributes.value: GSM3141939