title: GSM3074843: E11 pre-HSCs; Mus musculus; RNA-Seq
library_construction_protocol: YS were carefully collected and vitelline vessels were cut off. After removal of the head and tail, the blood was flushed away by injecting PBS/FCS/PS or antibodies with a glass capillary directly inside the aorta of the embryo trunk.  Aorta-Gonad-Mesonephros (AGM) regions were dissected, pooled and dissociated by collagenase treatment (0.125% collagenase type I in PBS/FCS/PS) for 1 hour at 37°C. After dissociation, cells were washed, stained with various antibodies. Cells were sorted with a FACS Aria III flow cytometer. Single whole (w)IAHCs were mechanically picked-up from live thick embryo slices by using a CellCelector mechanical cell-picker. Location (dorsal or ventral) of the picked wIAHC was recorded. Single cells were sorted in the wells of 96-well PCR plates, each well containing 100ml of TRIzol reagent and 0.2ul of a 1:250,000 dilution of ERCC spike ins (Thermo Fisher). wIAHCs were collected after pick-up in tubes containing the above solution. Total RNA was extracted from single cells, according to TRIzol manufacturer's instructions (Thermo Fisher) with few alterations. GlycoBlue reagent (0.2μl) was added to each sample to facilitate RNA precipitation. After overnight isopropanol precipitation, RNA pellets were air dried for up to 15 minutes, and suspended in CEL-Seq first-strand primer solution Single cells (or wIAHCs) RNA obtained after TRIzol extraction was suspended in primer solution, denatured at 70˚C for 2 minutes and quickly chilled. First strand synthesis was carried out at 42˚C for 1 hour followed by a second strand synthesis step at 16˚C for 1 hour. cDNA was cleaned-up and in vitro transcription was set-up overnight at 37˚C. Amplified RNA was fragmented and cleaned-up for 3' adapter ligation, reverse transcription reaction and PCR amplification. Libraries were sequenced with an Illumina NextSeq 2500 by using 75 bp paired end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3074843