GSM5089163: 156560262_3_D8_CAGAGAGG-CTAAGCCT_L008; Homo sapiens; RNA-Seq
Samples were single-cell sorted into 96-well PCR plates containing 5 µL of DNA Suspension Buffer (Teknova) with 1% BSA (Sigma-Aldrich) per well. Plates were sealed and briefly vortexed before being spun at 800 × g for 1 minute and immediately transferred to dry ice. Plates were stored at -80°C for at least overnight before proceeding. Once ready to proceed, the plates were thawed on ice and spun at 800 × g for 1 minute to collect all liquid. We used a modified version of a previously published method to amplify the whole transcriptome (8). In brief, the reverse transcriptase that is used to make cDNA has a terminal transferase activity. Then, a “template‐switch” primer incorporates a second universal priming sequence yielding cDNA with two universal priming sequences.  Custom primers were synthesized by IDT using sequences published by Trombetta, et al. (8). In brief, reverse transcription occurred in two steps.  The initial step was conducted in 5 µL of the lysed cell, 2.4 µM 3' SMART CDS Primer II A (IDT), 2.4 mM each dNTP mix (GeneAmp) and 6.32 U RNaseOUT (Invitrogen).  The samples were incubated at room temperature for 1 minute, 72°C for 3 minutes and held on ice before beginning the second step.  The next step was conducted with SuperScript II Reverse Transcriptase Kit (Invitrogen), using 7 µL of the step 1 product, 1× First Strand Buffer, 2.5 mM DTT, 110 U SuperScript II and an additional 11 U RNaseOUT, as well as 1 µM SMARTer II Oligonucleotide (IDT) and molecular-grade water to 11 µL. The samples were incubated at 42°C for 90 min, followed by a limited cycle amplification of 10 cycles at 50°C for 2 min and 42°C for 2 min, concluding with a final extension at 72°C for 15 minutes. Whole transcriptome amplification (WTA) immediately proceeded with the 11 µL of the reverse transcription product, using 1× KAPA HiFi HotStart Ready Mix (Roche) and 0.5 µM IS PCR Primer (IDT).  Amplification parameters were 98°C for 3 min and 22 cycles of 98°C for 15 sec, 67°C for 20 sec and 72°C for 6 min, followed by a final extension of 72°C for 5 min.  The WTA product was cleaned using AMPure XP beads (Beckman Coulter).  The PCR product was incubated at room temperature for 5 min with 0.8 volumes of XP beads before being placed on a magnet.  The beads were washed three times with 100 µL of 80% ethanol.  After briefly drying, the beads were resuspended in 20 µL of 1× TE Buffer (Sigma Aldrich) and 18 µL of supernatant were was transferred to a new 96-well PCR plate. Quality and quantitation were measured by TapeStation using a High Sensitivity D5000 ScreenTape (Agilent).  Samples with a concentration ≥200 pg/µL were carried forward for sequencing according to the Nextera XT DNA Library Preparation Kit (Illumina) protocol.  Samples were dual-indexed using the Nextera Index XT Kit (Illumina) and a scheme that would allow for eight pools without overlapping indices. After pooling, the concentration of each pool was measured by Qubit dsDNA HS Assay Kit (Invitrogen) and sizing was determined by Fragment Analyzer HS Small Fragment Kit (Agilent).  A smear analysis was conducted in the range of 150 to 1000 bp, and the average peak size along with the Qubit results were used to determine the molarity.  Each pool was then diluted to a concentration of 2 nM.  The samples were sequenced on the Illumina HiSeq 2500 using PE50 on a high-output 8-lane flow cell with one pool per lane.