NextSeq 500 paired end sequencing; RNA-seq as a global measure of the similarity between human pluripotent stem cell and fetal liver derived B cell hierarchies
RNA-seq as a global measure of the similarity between human pluripotent stem cell and fetal liver derived B cell hierarchies
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 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).
Experimental Factor: developmental stage
embryo
Experimental Factor: cell type
pro-B cell
Experimental Factor: cell line
H1-hESC
Experimental Factor: phenotype
IL7R+; wild type