A front limb of a quadruped. (The Random House College Dictionary, 1980)
Attributes by original data submitter
Sample
source_name
forelimb bud at E12.5
strain
C57BL/6
tissue
forelimb bud
age
E12.5
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
For sampling, mouse forelimb buds of each stage were dissected and treated with collagenase for 10 min at RT. Then, cells were dissociated into single-cell suspensions by pipetting and 40 μm mesh filter (Funakoshi, Cat. No. HT-AMS-14002), and frozen in CryoStor® media (Stemcell technologies Cat. No. ST07930) with Mr. Frosty™ (Thermo Scientific, Cat. No. 5100-0001) at −80°C for overnight Stored cells were melted in 38 °C water bus, and spun down at 500 g for 5min at 4 °C, which was followed by a wash using 50 μl of cold PBS once and centrifugation at 500g for 5 min. Ten thousands of cells per sample were collected without distinguishing dead cells, and lysed using 50 μl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1 % IGEPAL CA-630). Immediately after lysis, cells were spun at 1000 g for 10 min at 4 °C, and the supernatant was discarded. For transposition reaction, cells were re-suspended in the transposase reaction mix (25 μl 2× TD buffer, 2.5 μl transposase (Illumina) and 22.5 μl nuclease-free water), and incubated for 30 min at 37 °C. For control, 50 ng control genome DNA was also transposed. The reaction mix was purified using DNA Clean & Concentrator-5 (zymo, Cat. No. D4004) by adding 350 μl of DNA binding buffer, and eluted in 10 μl. After 5 cycle pre PCR amplification, the optimal number of PCR cycles was determined by a preliminary PCR using KAPA Library Amplification Kit (KAPA, Cat. No. KK2702) and estimated to be 4 cycles. The PCR products were purified using 1.8x volumes of Agencourt AMPure XP (Beckman Coulter, Cat. No. A63880).