title: GSM3526358: BaumsDatabaseID_12719; Acropora palmata; OTHER
library_construction_protocol: Coral tissue samples were snap frozen in liquid nitrogen and kept at -80° C before extraction using the DNeasy Blood & Tissue Kit (QIAGEN) with the following modifications.  Time of incubation in extraction buffer was increased to 16-20 hours. Further, only the second elution was retained for library production as this fraction contained the high molecular weight (HMW) DNA . A total of 100ng of HMW genomic DNA was digested with FspEI (NEB) at 37°C for 4 hours. Five µL of digested DNA was run on a 1% agarose gel to verify digestion. Ligation was performed in a 30 µL reaction with 20 µL of digested DNA, 0.2 µM each of two adaptors, 1 mM ATP and 800 U of T4 DNA ligase (NEB). Ligation was carried out at 4° C overnight. Adaptor and primer sequences are provided in the supplementary material (Table S3). Ligation products were amplified in 40 µL reactions containing 15 µL of ligated product, 0.2 mM of each primer (p1 and p2), 0.3 mM dNTP, 1X Phusion HF buffer and 0.4 U Phusion high-fidelity DNA polymerase (NEB). PCR was performed in an Eppendorf Mastercycler with 22 cycles of 98°C for 5 s, 60°C for 20 s, 72°C for 10 s, and a final extension of 5 min at 72°C. The target DNA band (approx. 100 bp) was excised from a 10% TBE polyacrylamide gel, and the DNA was diffused from the gel in 50 µL of nuclease-free water for 12 h at 4°C. Barcodes were added in a second PCR amplification (total volume =20 µL), which contained 7 µL of gel-extracted PCR product, 0.2 mM of each primer (p3 and index primer), 0.3 mM dNTP, 1X Phusion HF buffer and 0.4 U Phusion high-fidelity DNA polymerase (NEB). Seven PCR cycles with the same profile outlined above were performed. PCR products were purified using QIAquick minElute PCR purification kit (Qiagen) and then with Ampure XP beads (Agencourt). Libraries were run on an Illumina HiSeq 2500 sequencer using 50 nt single read sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3526358