title: GSM4479022: GC059869_C08; Mus musculus; RNA-Seq
library_construction_protocol: Samples were incubated in 250µl of 2% FBS/PBS with fluorochrome-conjugated primary antibodies for 30 minutes, with shaking every 10 minutes. Primary antibodies were washed with 2% FBS/PBS, and cells were resuspended in 2.5mg/ml DAPI (Invitrogen D1306) 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, cloneAPA5, eBiosciences), PE-Cy7-conjugated anti-CD24 (1:100, clone M1/69, BD), FITC-conjugated anti-CD29 (1:100, clone Ha2/5, BD Biosciences),APC-Cy7-conjugated anti Epcam (1:100, clone G8.8, Biolegend). Data analysis andcell sorting were performed on a FACSAria sorter using the FACS DiVa software (BDBiosciences). Dead cells were excluded with DAPI; CD45+, CD31+ and CD140a+ cells excluded (Lin+) before analysis of the Tomato+ cells scRNA-seq was performed using a modified Smartseq-2 protocol. 1µL lysis buffer in384 well PCR plates for cell sorting was prepared with 0.4% v/v Triton-X lysis buffer,2.5mM dNTPs and 2.5µM oligo-dT30-VN. Reverse transcription and PCR was performed according to the Smartseq-2 protocol with reduced volumes: 1µl of reverse241 transcription mix instead of 5.7µl and 3µl PCR Master mix instead of 15µl. cDNA was amplified for 23 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. 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 12 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 HiSeq4000 with 1x51bp read length
attributes.tag: GEO Accession
attributes.value: GSM4479022