alias: GSM3289036
title: GSM3289036: BFUEs_Day3_93; Mus musculus; RNA-Seq
library_construction_protocol: 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) cell culture protocol: BFU-E cell culture. FACS-isolated BFU-E enriched cells were cultured for the denoted number of days in progenitor culture media (PCM) consisting of serum free expansion media (SFEM2, Stem Cell Technologies) supplemented with 100 ng/mL murine SCF (Peprotech), 40 ng/mL murine insulin-like growth factor 1 (Peprotech), 2 U/mL human erythropoietin (Amgen), 1% penicillin/streptomycin (Gibco), and for indicated cultures 100 nM Dexamethasone (Sigma).   Single cell transcriptome library preparation.  SmartSeq2 library prep was performed by depositing single cells in PCR plate wells by FACS.  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).
identifiers.namespace: GEO
identifiers.id: GSM3289036
attributes.tag: GEO Accession
attributes.value: GSM3289036