alias: GSM2988266
title: GSM2988266: Single Embryonal Progenitor Cell TAAGGCGA-GCGTAAGA; Mus musculus; RNA-Seq
library_construction_protocol: Samples were incubated in 250μl of 2% FBS/PBS with fluorochrome-conjugated primary antibodies for 30min, with shaking every 10min. Primary antibodies were washed with 2% FBS/PBS, and cells were resuspended in 2.5mg/ml DAPI (Invitrogen) before analysis. The following primary antibodies were used: APC-conjugated anti-CD45 (1:100, clone 30-F11, eBiosciences), APC-conjugated anti-CD31 (1:100, clone 390, eBiosciences), APC- conjugated anti-CD140a (1:100, clone APA5, eBiosciences) and PE-conjugated anti- CD49f (1:200, clone GoH3, eBiosciences) for embryos; PECy7-conjugated anti- CD24 (1:100, clone M1/69, BD Biosciences), APC-conjugated anti-CD29 (1:100, clone eBioHMb1-1, eBiosciences), PE-conjugated anti-CD45 (1:100, clone 30-F11, eBiosciences), PE-conjugated anti-CD31 (1:100, clone MEC 13.33, BD Biosciences), PE-conjugated anti-CD140a (1:100, clone APA5, eBiosciences) for adult MGs. Data analysis and cell sorting was performed on a FACSAria sorter using the FACS DiVa software (BD Biosciences). Dead cells were excluded with DAPI; CD45+, CD31+ and CD140a+ cells were excluded (Lin+) before analysis of the GFP+ cells. Single cell transcriptomes were generated using a modified Smartseq-2 protocol74. 1μL lysis buffer in 384 well PCR plates for cell sorting was prepared with 0.4% v/v Triton-X lysis buffer, 2.5mM dNTPs, 2.5μM oligo-dT30-VN and ERCC controls at a final dilution of 1:100 million. Reverse transcription and PCR was performed according to the Smartseq-2 protocol with reduced volumes: 1μl of reverse B372transcription mix instead of 5.7μl and 3μl PCR Master mix instead of 12.5μl. cDNA was amplified for 24 cycles and cleaned using HighPrep PCR beads (MagBio Genomics) at a 0.8x ratio on a Hamilton STAR (Hamilton Germany GmbH) liquid handler, eluted in 30μl buffer EB (Qiagen) and transferred to 384_PP acoustic plates (LabCyte). DNA quantification was performed with Picogreen assay (Thermofisher) and a subset of samples were selected for quality control using a Agilent 2100 BioAnalyser (Agilent Technologies) using a High Sensitivity DNA kit. After initial quality control 24 samples were discarded with cDNA yields of less than 21ng. These samples were replaced with 24 cells from another 384 well plate from the same cell sort by plate reformatting with an acoustic dispenser (LabCyte Echo 525). Next generation sequencing library preparation was performed using a Nextera XT DNA library preparation kit with volumes reduced by one-tenth (Illumina) using an acoustic dispenser. In brief, 100pg of cDNA in a volume of 500nL was tagmented by adding 1.5μl Tn5-buffer mix and incubating for 10 min at 55°C. Tagmented samples were barcoded with Nextera index sets A - D and amplified with 11 cycles of PCR. After PCR, all samples were pooled and cleaned using HighPrep PCR beads at a 0.6x ratio. Library pools were eluted in buffer EB and quality control performed using an Agilent 2100 BioAnalyser and High Sensitiviy DNA chip before adjusting to a concentration of 4nM. The diluted pools were quantified using a KAPA qPCR library quantification kit on a LightCycler 480 (Roche) before a final dilution to 2nM. The pool of 384 samples was sequenced on 2 lanes of a HiSeq2500 in high output mode v4 chemistry with 1x100bp read length. scRNAseq
identifiers.namespace: GEO
identifiers.id: GSM2988266