title: GSM8829595: WT_D6_1_sp; Homo sapiens; RNA-Seq
library_name: GSM8829595
library_construction_protocol: For RNA-seq: WT and GPX2 stem cells derived foregut endoderm and WT, GPX2 KO, WT treated with H2O2 (10nM) and GPX2 KO treated with sodium selenite (100pM) definitive endoderm cells differenced spontaneously for additional 5 days were subjected to RNA-seq.  Total RNA was extracted and purified using Trizol as previously described (Varshney et al., 2017). RNAseq was performed according to the TruSeq RNA Library Prep Kit protocol. For ATAC-seq: The WT, GPX2 KO definitive endoderm cells differented spontanously  for additional 5 days were subjected to ATAC-seq. Cells  were dispersed to single cells using TrypLE. The 5 x 104 cells were resuspended in 50 µl of cold ATAC-RSB buffer (10mM Tris-HCl pH = 7.4, 10 mM NaCl, 3 mM MgCl2,, 0.1% Tween-20, 0.1% NP-40). For nuclei extraction, the cells were incubated with 0.1% digitonin for 3 min at 4 °C and centrifuged at 500 x g for 10 min at 4 °C with the addition of 1 ml ATAC-RSB. The supernatant was discarded, and nuclei were prepared for the transposition reaction. The solution containing transposed fragments was purified using the Zymo DNA Clean & Concentrator (Zymo Research, USA) and the transposed DNA was eluted in 20 μl H2O and stored in -20 °C. Barcoded adapters were added to the transposed fragments by PCR with the NEBNext Ultra II Q5 2x Master Mix (New England Biolabs, USA), according to the producer protocol and after purified with the AMPure XP beads. The samples were 150 bp paired-end sequenced on the NovaSeqX platform. For scRNA-seq: The WT and GPX2 KO stem cells derived beta cells or early pancreatic progenitors cells were dispersed with Tripsin, washed with 0.1% saponin – 1% BSA – PBS, filtered through 30 μm mesh filter. Afterwards cells were counted using Countess and adjusted to 700-1,200 cells/μl. scRNA-seq was performed with 10xGenomics platform or ScaleBio and Illumina according to the manufacturer's protocol. Libraires were pair-end sequenced with depth of 40,000 reads per cell. paired-end libraries
identifiers.namespace: GEO
identifiers.id: GSM8829595_r1