title: GSM2594182: Primary erythroid cells, growth protocol 2_ATAC; Homo sapiens; ATAC-seq
library_construction_protocol: Human: Primary erythroid stem cell progenitors were isolated from peripheral blood, using CD34 coupled magnetic beads. Cells were expanded for 7days in low Epo conditions (0.5 IU/ml)) and then transfered for differentiation in high Epo (3.0 IU/ml) medium. On day 10, cells were washed in cold PBS, and counted.  70,000  cells were used for the ATAC-seq protocol. Nuclei were isolated by lysing the cells and transposition with Tn5 transposase, DNA extraction and sequencing was performed as published by Buenostro et al 2013. ATAC-seq was performed as previously published (Buenrostro:2013). Nuclei of 70000 lysed cells were isolated and transposition with Tn5 transposase (Nextera, Illumina) was performed for 30 minutes at 37 °C. The DNA was extracted using a MinElute kit (Qiagen). Libraries were then amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom ATAC-seq primers published by Buenrostro and colleagues. ATAC-seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent) and quantified using the universal library quantification kit (KAPA Biosystems). For the ATAC-seq background 100 ng of genomic DNA was incubated with the Tn5 transposase and the library was made following the protocol described before. The strategy was to isolate open chromatin fragments specifically as they underly regulatory genomic regions. These are short stretches of DNA, with low abundance in the genome and thus require to be processed efficiently. The fragments can then be multiplexed and sequenced.
attributes.tag: GEO Accession
attributes.value: GSM2594182