title: GSM1477981: Chicken Embryonic Fibroblasts; Gallus gallus; ncRNA-Seq
library_construction_protocol: Blastodermal cells at Eyal-Giladi and Kochav (EG&K) stage X were collected by gentle dissociation of stage X blastoderms (n = 30) from freshly laid eggs in phosphate buffered saline (PBS) followed by centrifugation at 1,250 rpm for 10 min. Fertilized eggs were incubated at 37.5°C under 50–60% relative humidity. PGCs and GSCs were isolated from the gonads of chicken embryos at E6.0 (mixed sex, n = ~2000) using the magnetic-activated cell-sorter (MACS) method. Gonads were dissociated by gentle pipetting in 0.25% trypsin and 0.05% ethylenediaminetetraacetic acid (EDTA). After adding 10% fetal bovine serum (FBS) to inactivate the trypsin-EDTA and briefly centrifuging at 200 × g for 5 min, total gonadal cells were incubated with anti-stage specific embryonic antigen (SSEA-1) for 20 min at room temperature. Cells were washed with 1 mL MACS buffer (0.5% BSA and 2 mM EDTA in PBS), and the supernatant was completely removed by centrifugation. The pellet was mixed with 100 μL MACS buffer supplemented with 20 μL goat anti-mouse IgM microbeads for 15 min at 4°C. Cells were washed with 500 μL MACS buffer and loaded onto a MACS column (Miltenyi Biotec GmbH). After several batches of cell preparation, the PGC-enriched fraction and GSCs were separated. The survival rate of PGCs purified by MACS were >95%. CEFs were collected by dissociating the embryonic body (E6.0, n = 6) in 0.25% EDTA at 37°C for 20 min. Cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic–antimycotic in a 5% CO2 atmosphere at 37°C. Approximately 5 µg total RNA from each test sample (PGCs, stage X blastoderms, GSCs, and CEFs) was used to generate high-throughput NGS data using the Illumina HiSeq (Beijing Genomics Institute, China) following standard protocols from Insilicogen, Korea
attributes.tag: GEO Accession
attributes.value: GSM1477981