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 Eppendorf tubes containing RLT buffer (QIAGEN) with 1% β-mercaptoethanol (Sigma Aldrich) according to their GFP fluorescence intensity and cell cycle category (Sony MA900 sorter). Total RNA was subjected to a modified version of SMARTseq-2 protocol published by Picelli et al. 2013 (doi: 10.1038/nmeth.2639).In short, total RNA was isolated with RNeasy Micro kit (Qiagen) and eluted in nuclease free water. The RNA was desiccated to 3 µl (in a Eppendorf Concentator Plus for 25 min at room temperature) and subjected to a primer mix (final concentration: 2.4 mM dNTP (Invitrogen), 240 nM dT-primer* and 4 U RNase Inhibitor (NEB)). RNA is denatured (3 minutes, 72 °C), followed by reverse transcription (42°C, 90 min + 70 °C 15 min.) after filling up to 10 µl with RT buffer mix (1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer*, 9 U RNase Inhibitor and 90 U Superscript II). Optimal number of cycles for amplification is determined by qPCR. Thus, the cDNA is amplified using 1x Kapa HiFi HotStart Readymix (Roche) and 250 nM UP-primer* under following cycling conditions: 98°C for 3 min, 14 or 16 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 0.6x volume of Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5 % (w/v) PEG 8000 and 2 M sodium chloride solution and eluted in 12 ul nuclease free water. The cDNA quality and concentration is determined with the Fragment Analyzer (Agilent). For library preparation 2 µl cDNA was tagmented using 0.8 µl bead-linked transposome and 1x Tagmentation Buffer 1 (Illumina DNA Prep, (M) Tagmentation, Illumina) at 55 °C for 15 min. The reaction was stopped with 0.1 % SDS (37°C, 15 min). The magnetic beads in the reaction tubes are bound to a magnet and the supernatant is removed. The beads are resuspended with 1x concentrated KAPA Hifi HotStart Ready Mix and 300 nM dual indexing primers and subjected to a PCR (72 °C 3 min, 98 °C 30 sec, 12 cycles [98 °C 10 sec, 63 °C 20 sec, 72 °C 1 min], 72°C 5 min). Libraries are purified with 0.9x volume Sera-Mag SpeedBeads, followed by a double size selection with 0.6x and 0.9x volume of beads.
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: SAMEA11319829
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-11210:Sample 13
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell cycle stage
attributes.value: G1
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: Stage 4 Day 1
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: number pairs
attributes.value: 62481600
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: phenotype
attributes.value: GFP negative
attributes.tag: progenitor cell type
attributes.value: human embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-11210:Sample 13