GSM5768746: NB4; Homo sapiens; RNA-Seq
Following surgical resection of peripheral neuroblastic tumors, fresh tissue samples were immediately transferred to DMEM/F12 medium (MULTICELL), supplemented with 2% FBS (Gemini Bio-Products) and were delivered on ice within 60 minutes to the laboratory to be processed. Tissue processing was completed within 90 minutes of collection. Samples were first rinsed in ice cold PBS (MULTICELL) with 2% penicillin-streptomycin (BBI life science) and then minced into 1mm3 pieces using curved scissors under sterile conditions. The fragments were further enzymatically dissociated into single cells using LiberaseTM (Roche) with a final concentration of 0.26 U/mL and a final volume of 25ml Liberase solution/0.5g tissue on a shaker with a speed of 85 rpm for 45 min at 37℃. The Liberase was diluted in Hyclone L-15 Leibovitz media. DMEM/F12 media supplemented with 10% FBS was used to stop dissociation. The resulting single-cell suspension was filtered through a 70µm nylon cell strainer (BD Falcon) and then centrifuged for 5 min at 300g at room temperature. The cell pellet was resuspended in 500µL of PBS supplemented with 0.1% BSA (BBI life science) and passed through a 40µm nylon cell strainer (BIOFIL). This cell suspension was stained with Calcein-Blue (Invitrogen) and DRAQ5 (CST) prior to fluorescence activated cell sorting (FACS) in order to isolate live and nucleated cells (Calcein-Blue+ DRAQ+). Single cells were sorted using a BD Becton Dickinson FACSAriaII into 96-well PCR plates. Each well of the 96-well plate contained 3µL lysis buffer (10U Recombinant RNase Inhibitor (Takara Bio), 0.2% Triton X-100 (Sigma- Aldrich), 3mM dNTP mix (Takara Bio)), and reverse transcription reagents (see below). Immediately following FACS, plates were briefly centrifuged and stored at -80℃. scRNA-seq was conducted using the Smart-seq2 protocol with some modifications being made to incorporate unique molecular identifiers (UMIs) into the 3' end of transcripts. Reverse transcription was performed directly in the 96-well plates by incubation at 72℃ for 5 min, after which the plate was replaced on ice to allow the oligo-DT primer to hybridize to the poly(A) tail of the mRNA molecules. The oligo(dT) primer used in reverse transcription included an additional 8bp cell barcode, 9bp unique molecular identifiers (UMIs) and template-switching oligo sequence. PCR amplification was performed by adding 15µL PCR mix containing 0.5U KAPA HiFi HotStart (Kapa Biosystems), 1x KAPA Buffer (Kapa Biosystems), 12.5mM MgCl2 (Kapa Biosystems), 5µM ISPCR Primer and 7.5mM dNTP mix (Takara Bio). PCR amplification was performed in a thermal cycler (BIORAD C1000 Touch Thermal Cycler) at 98℃ for 3 min, 24 cycles of 98℃ for 20s, 67℃ for 15s, and 72℃ for 6 min, and a final incubation at 72℃ for 5 min. PCR products were purified using 1X AMPure XP bead (Beckman, Cat. #A63882) and Qubit dsDNA HS Assay Kit (Invitrogen, Cat. #Q32854). TN5 tagmentation and library amplification realized 3'end fragments by Nextera XT DNA Sample Preparation Kit (Illumina, Cat # FC- 131-1024) according to the manufacturer's instructions while P5_TSO and Nextera_N7xx took the place of the custom primers. Pooled single cell libraries were sequenced at an average depth of 0.5 million reads per cell on an Illumina HiSeqX10 instrument using a 2 x 150bp paired end sequencing kit (Genewiz). scRNA-seq of primary neuroblastic tumor using the Smartseq2-UMI platform. OTHER: Modified Smart-seq2