GSM3289334: NoDex_Pair4_Cell1; Mus musculus; RNA-Seq
cell extraction protocol: Erythroid progenitor cell isolation. Fetal liver was dissected from embryonic day 14.5 mice and disrupted and homogenized with manual pipetting, followed by lysis of mature red blood cells with ammonium chloride solution (Stem Cell Technologies).  Remaining fetal liver cells were stained with biotinylated antibodies against the following surface proteins: CD3e, CD45R, Ly-6G, CD11b, Ter119 (all 5 from the BD-Pharmingen biotin mouse linage depletion panel), Ly-6A/E (i.e. Sca-1, cloned D7, E-Biosciences), CD16/32 (clone 93, E-Biosciences), CD41 (clone EbioMWReg30, E-Biosciences), and Ter119 (E-biosciences). Cells were then incubated with streptavidin-labeled magnetic beads (BD) to deplete cells labeled with biotinylated antibodies.  Remaining unlabeled cells were then purified by FACS (staining procedure described below) on a FACS Aria sorter (BD). FACS selection protocol: Antibody staining and FACS.For FACS purification of erythroid progenitors, lineage-depleted fetal liver cells were stained with the following: Propidium Iodide (Sigma), APC or PE-Cy7 anti-CD117 (clone ACK2, E-Biosciences), PE anti-CD71 (clone R127217, E-Biosciences), PE anti-CD24 (clone M1/69, E-biosciences), FITC streptavidin (E-Biosciences).  For quantification of erythroid differentiation markers, cultured BFU-E cells were stained with PE anti-CD71 (clone R127217, E-Biosciences) and APC anti-Ter119 (E-Biosciences).  Flow cytometry for erythroid differentiation markers was performed on a FACS Fortessa cytometer (BD) daughter cell isolation protocol : Single BFU-E cells were FACS-deposited into individual wells of a 96-well plate containing PCM either with or without 100 nM dexamethasone.  Plates were briefly spun down and visualized by light microscopy using a Zeiss Primovert microscope to confirm the wells with only a single cell. Those wells were further assessed 16 hours later to identify the cells that completed a single cell division. Cells from wells that contained a single pair of daughter cells were removed from the 96-well plate by mouth pipetting, using hand-drawn glass capillary pipettes (Drummond Scientific) and a Nikon SMZ 1500 microscope. Both daughter cells were deposited in a 30 uL drop of 2% FBS (Gibco) in PBS and the two cells were manually separated by mouth pipetting.  Each individual daughter was then mouth pipetted into SmartSeq2 lysis buffer to perform single cell transcriptome profiling with single cell RNA sequencing. Single cell transcriptome library preparation.  Each individual daughter was then mouth pipetted into SmartSeq2 lysis buffer to perform single cell transcriptome profiling with single cell RNA sequencing.  Single cells were lysed in TCL buffer (Qiagen), containing RNase inhibitor (Clontech), Oligo-dT (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3', IDT), and dNTPs (Invitrogen).  Lysates were then reverse transcribed at 42 deg C with SuperScript II reverse transcriptase (Invitrogen) in the presence of First-Strand Buffer (Invitrogen) containing DTT (Invitrogen), betaine (Sigma), magnesium chloride (Sigma), and template switching oligonucleotide (5' -AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' where rG = riboguanosine, and +G = LNA-modified guanosine, Exiqon/Qiagen).  Reverse-transcribed products were then amplified using Kapa HiFi Hotstart PCR master mix (Kapa) and an IS PCR oligo primer (5' -AAGCAGTGGTATCAACGCAGAGT-3', IDT). Amplified double-stranded cDNA libraries were then purified using Agencourt Ampure XP beads (Beckman Coulter), followed by DNA concentration quantification using the Qubit dsDNA HS kit (Life Technologies).  0.2 ng of double-stranded cDNA was used in a tagmentation reaction using the Nextera XT DNA sample preparation kit (Illumina) per manufacturers protocol. Tagmented transcriptomes were then indexed using the Nextera XT 96-index kit for 384 samples (Illumina) per manufacturers instructions, and samples were then sequenced on a HiSeq 2500 sequencer (Illumina).