GSM8647871: attB/P_mESC_K562_Lane1_GEX; Homo sapiens; RNA-Seq
For loxPsym+ mESC scRNA-seq experiment, 300,000 parental cells were transfected in 6-well plates as described above with 1 μg of Cre and 200 ng of the reporter per transfection. 10 individual wells were transfected. At 24 hours post transfection, 5 wells were treated with 20 μM Pifithrin-α for 48 hours total. At 72 hours post transfection, both Pifithrin-α treated and untreated cells were harvested and approximately 500,000 RFP positive cells were FACS sorted into a combined tube based on the activity of the Cre reporter. Untransfected parental cells were harvested, and 1 million cells were used as input in parallel with Cre-sorted cells for the protocol.  For the attB/P+ K562 and attB/P+ mESC “Lane 1” experiment, 200,000 parental cells were nucleofected (2 reactions) or transfected (3 reactions) with 1 μg of Bxb1 plasmid as described above. All cells were harvested at day 3 post-transfection and used as input for the protocol. For the “Lane 2” experiment, attB/P+ K562 populations bottlenecked to 1000 cells after Bxb1 treatment were thawed and split once before being used as input in the protocol. For both Lanes, 500,000 cells from each population were mixed and were used for the subsequent steps.  After washing twice with cold 1X PBS (Gibco), 1 million cells per sample were resuspended in 400 μl of cold PBS. Cells were fixed with 1600 μl of cold 100% methanol, added dropwise with swirling. Cells were left on ice to fix with gentle swirling to mix every 5 minutes during the incubation. Cells were rehydrated with 4 mL of cold 1X PBS added slowly with gentle swirling of the tube. Cells were spun down and resuspended in 60 μl of PBS. For the loxPsym+ mESC experiment, approximately 60,000 cells and 380,000 cells in total were counted in the Cre and parental samples respectively using a Countess automated cell counter (Thermo) of which all and 100,000 cells in 18 μl of PBS were used for T7 IVT respectively. For the attB/P+ K562/mESC experiment, approximately 600,000 and 450,000 cells were counted of which, 100,000 cells per sample in 18 μl of PBS were used for T7 IVT. IVT reactions were set up in 30 μl total volume with the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S) with 2 μl of each NTP, buffer and enzyme. Reactions were incubated at 37°C for 1 hour in a thermocycler. Cells were immediately moved to ice and 20 μl of cold PBS was added to each sample. Approximately 40,000 cells were processed per lane of a 10X Genomics Single Cell 3' HT with Feature Barcoding kit. Transcriptome libraries were prepared as per the manufacturer's protocol. To prepare libraries from T7 transcripts captured using CS1 and CS2, we started with the supernatant from the clean-up after cDNA amplification of the standard 10X Genomics feature barcoding protocol for the loxPsym+ mESC experiment. For the attB/P+ K562/mESC experiment, we spiked in two construct specific primers (oSP1059, oSP1060) at 0.5 μM into the cDNA amplification step. Two rounds of PCR were performed with primers specific to the shuffle cassette. In PCR1, oSP997 (CS1-TruSeq2), oSP1022 (CS2-TruSeq2) and oSP1061 (feature-cDNA primer F) primers were used (1.25 μl of 10 μM each) in a 50 μl Q5 polymerase reaction with 5 μl of template. Cycling conditions: 98°C - 45 s; cycles of 98°C - 20s, 60°C - 5s, 72°C - 5s; 72°C - 60s, 4°C - hold. 15 cycles of PCR1 were performed for the loxPsym+ mESC experiment and 12 cycles for the attB/P+ K562/mESC experiment. loxPsym+ mESC  were cleaned up with AmpureXP beads (1X) and eluted in 15 μl of Qiagen buffer EB. attB/P+ K562/mESC reactions were cleaned up with AmpureXP beads (1X) and eluted in 30 ul of buffer EB. In PCR2, 5 μl of PCR1 eluate was used as input into either one or two (loxPsym+ mESC experiment)  50 μl Q5 Polymerase reactions per sample with sample index primers (1.25 μl of 10 μM each) that add Illumina adapters. Reaction progress was monitored using SYBR green and was stopped before saturation. Cycling conditions for the loxPsym+ mESC experiment: 98°C - 5 min; 9 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s; 72°C - 60s, 4°C - hold. Cycling conditions for the attB/P+ K562/mESC experiment: 98°C - 1 min; 8 cycles of 98°C - 20s, 63°C - 20s, 72°C - 1min; 72°C - 60s, 4°C - hold. After a clean up with AmpureXP beads (1X), quality of the libraries was confirmed (prominent single-peak) by running them on a TapeStation D1000 ScreenTape (Agilent).  Primer sequences:  oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC oSP1061_FeaturecDNA_Primer1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG oSP1059_CS2_SpikeIn GGGGGGTTTTTTTTTTCCTTAGCC oSP1060_CS1_SpikeIn GGGGGGTTTTTTTTTTGCTAGGAC