organism: Homo sapiens
description: Protocols: hESC-derived pancreatic cells at Stage 4 Day 1 of the pancreatic differentiation protocol (Rezania et al., 2014; Petersen et al., 2017) were dissociated into single cells with TrypLE and resuspended into a FACS buffer containing 0.5% BSA and 2 mM EDTA (Thermo Fisher) in 1X PBS. The cells were index-sorted according to their GFP expression into 384-well plates on a BD FACSAria and snap-frozen. Cells are processed with SMARTseq-2 protocol miniaturized and modified based on Picelli et al. 2013 (doi: 10.1038/nmeth.2639). In short, cells are sorted in 0.5 µl 0.2% Triton X-100 solution with RNase Inhibitor into a 384-well plate, frozen at -80°C. After thawing, the cell lysate is mixed with 0.5 ul of a primer mix (containing 5 mM dNTP (Invitrogen), 0.5 uM dT-primer*, 1 U RNase Inhibitor (NEB)). RNA is denatured (3 minutes 72 °C), followed by reverse transcription (42 °C 90 min + 70°C 15 min) after adding 1.5 ul RT buffer mix (1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 uM TSO-primer*, 2.3 U RNase Inhibitor and 25 U Superscript II). The cDNA is amplified using 1x Kapa HiFi HotStart Readymix (Roche) and 0.1 uM UP-primer under following cycling conditions: 98°C for 3 min, 23 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA is purified using 1x volume of Sera-Mag SpeedBeads (GE Healthcare) and eluted in 12 ul nuclease free water. For library preparation 1.5 µl cDNA is desiccated and rehydrated in 1 ul Tagmentation mix (1x TTBL buffer, 0.1 ul Tagment DNA Enzyme; from TruePrep DNA Library Prep Kit V2 for Illumina; Vazyme) and tagmented (55°C, 5 min), followed by an index PCR with 1x concentrated KAPA Hifi HotStart Ready Mix and 0.7 µM dual indexing primers (72°C 3 min, 98°C 30 sec, 13 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min). After PCR, libraries are quantified with AccuBlue Broad range chemistry (Biotium), equimolarly pooled and purified twice with 0.9x volume Sera-Mag SpeedBeads.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA11320513
attributes.tag: INSDC center alias
attributes.value: The Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
attributes.tag: INSDC center name
attributes.value: The Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:32Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:32Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11208:BSBTold1_02_J05
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: H1-hESC
attributes.tag: cell_type
attributes.value: pancreatic cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS
attributes.tag: gfp mean
attributes.value: 555
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: number of reads
attributes.value: L40657_Track-79984: 185889; L40657_Track-79354: 369442
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: phenotype
attributes.value: GFP low
attributes.tag: plate id
attributes.value: plate 1
attributes.tag: progenitor cell type
attributes.value: human embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-11208:BSBTold1_02_J05
attributes.tag: single cell well quality
attributes.value: OK
attributes.tag: time
attributes.value: Stage 4 Day 1
attributes.tag: well
attributes.value: J05