A front limb of a quadruped. (The Random House College Dictionary, 1980)
Attributes by original data submitter
Sample
source_name
Forelimb
strain
ICR
developmental stage
E12.5
tissue
Forelimb
chip antibody
rabbit anti-H3K4me3 (Abcam, ab1342)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Limb biopsies from embryos were cross-linked for 20 min in freshly made fixation buffer (1% formaldehyde 100 mM NaCl, 0.5 mM EGTA, 50 mM HEPES, pH 8.0) and quenched by the addition of glycine (125 mM). Tissue was washed twice with ice cold phosphate-buffered saline and snap-frozen in liquid nitrogen prior to sonication (14 cycles of 10 sec at 20% output with 60 sec on ice between cycles, Branson 450 sonicator) in lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0, protease inhibitors) to achieve an average sheared fragment size of 300 bp, with the primary smear between 200–450 bp. This sheared chromatin was used for immunoprecipitation. Single-end libraries were generated using the Illumina TruSeq ChIP seq library prep kit, with the only protocol modification being size selection after fragment amplification. The library size selected was 200–300 bp in length, and after gel purification, Agilent Bioanalyzer was used to confirm size. Libraries were then loaded on Illumina HiSeq 2000 and sequenced to generate 51 bp reads.