organism: Homo sapiens
description: Protocols: MIFF3 IPS (https://hpscreg.eu/cell-line/UOSi001-B) and H1 ES cells were cultured on matrigel-coated (Corning) 6 well plates in either mTeSR™1 (Stem Cell Technologies) or StemFit®AK (Ajinomoto Co., Inc, Tokyo, Japan). Cultures were passaged every 4-7 days using gentle dissociation reagent (SCT) according to manufacturer's instructions in the presence of Y27632 rock inhibitor. For these studies we used a human induced PSC (hIPSC) line, MIFF3, and corroborated our findings using the H1 human embryonic stem cell (hESC) line. An OP9/MS5 sequential co-culture system was used to differentiate hPSCs to the B cell lineage MIFF3 IPS cells were transformed with an ETV6-RUNX1 knock-in construct 22-400 cells were directly sorted into 800μl Trizol® and frozen at -80oC. RNA was extracted in the aqueous phase after addition of 160ul chloroform, precipitated with an equal volume of isopropanol supplemented with 20μg linear polyacrylamide and washed once in fresh 80% ethanol. The RNA pellet was re-suspended in 3μl water at 50oC for 10 minutes and then kept on ice. 100pg (or 1μl of stock if unquantifiable) of RNA used for first strand synthesis using SMARTER v3 (Clontech) before 16 cycles of amplification according to manufacturer's instructions. cDNA was purified on Agencourt ®AMPure®XP magnetic beads, washed twice with fresh 80% ethanol and eluted in 17μl elution buffer. 1μl cDNA was checked and quantified on an Agilent Bioanalyser high sensitivity DNA chip. Sequencing libraries were produced using Illumina® Nextera XT tagmentation according to manufacturer's instructions except using 150pg input cDNA, 5mins tagmentation and amplification 12 cycles using Illumina® XT 24 index primer kit. Libraries were cleaned using an equal volume (50μl) Agencourt ®AMPure®XP magnetic beads and re-suspended in 20μl elution buffer. Libraries were checked and quantified on an Agilent Bioanalyser high sensitivity DNA chip (size range 150-2000bp) and by Qubit® dsDNA BR (Molecular Probes).
attributes.tag: ENA first public
attributes.value: 2018-02-08
attributes.tag: ENA last update
attributes.value: 2018-01-29
attributes.tag: External Id
attributes.value: SAMEA104554848
attributes.tag: INSDC center alias
attributes.value: UCL Cancer Institute
attributes.tag: INSDC center name
attributes.value: UCL Cancer Institute
attributes.tag: INSDC first public
attributes.value: 2018-02-08T17:01:50Z
attributes.tag: INSDC last update
attributes.value: 2018-01-29T15:52:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6382:Sample_33
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MIFF3
attributes.tag: cell type
attributes.value: B cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: ETV6-RUNX1
attributes.tag: organism part
attributes.value: skin of prepuce of penis
attributes.tag: phenotype
attributes.value: LIN-CD34+CD45RA-; RUNX1 overexpression
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-6382:Sample_33