GSM9724062: E9.5 head, chimera 3; Mus musculus; RNA-Seq
We used a “cold digestion” protocol to maximize the cell viability after single cell dissociation. Embryos were dissected in a Petri dish filled with ice-cold HBSS (Sigma-Aldrich, 55037C-1000ML). A papain based Neural Tissue Dissociation kit (Miltenyi Biotec, 130-092-628) was used. All incubation steps were performed at 10°C in a pre-chilled refrigerated centrifuge. Each embryo was incubated with 500μL Mix1 for 15 minutes at 10°C. Mix 2 was added and embryos were further incubated for 20 minutes at 10°C. Embryos were pipetted with a P1000 pipette every 10 minutes until fully dissociated. Dissociated single cells were filtered through a 100μm strainer and spun down at 500 g for 5 min at 4°C. The supernatant was aspirated, and the cell pellet was washed twice by resuspending in 5mL FACS buffer (PBS with 2% FBS plus 0.1% Pluronic F-68). The cells were centrifuged again at 500g for 5min at 4°C. After the final wash, the cells were resuspended in FACS buffer for flow cytometry analysis or scRNA-seq with index sorting. Single cell capture and RNA extraction scRNA-seq with index sorting was performed as previously described50. Briefly, 96-well plates were prepared with 2 μL lysis buffer per well (1 U/μL RNase inhibitor (Clontech, 2313B), 0.1% Triton (ThermoFisher Scientific, 85111), 2.5 mM dNTP (Thermo Fisher Scientific, 10297018), 2.5 μM oligo dT30VN (Integrated DNA Technologies, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3'), and 1:600,000 ERCC (external RNA controls consortium) RNA spike-in mix at 1:600,000 (Thermo Fisher Scientific, 4456739) in UltraPure water (ThermoFisher Scientific, 10977015))101, which were stored at −80°C until use. Embryos were dissociated into a single cell suspension and stained for FACS as described above. Cells were sorted directly into lysis buffer plates on “single cell” precision mode with index sorting turned on. After a cell was sorted into each well, plates were centrifuged at 3000×g for 30 seconds at 4°C, immediately snap frozen on dry ice, then stored at −80°C.  Reverse transcription and pre-amplification Reverse transcription (RT) and cDNA pre-amplification were carried out with a modified Smart-seq3 protocol49. First, lysis buffer plates were first incubated at 72°C for 3 minutes, then immediately snap chilled on ice in order to anneal the oligo dT30VN primer.  For RT, 3 μL of RT mix was added to each well (25 mM Tris-HCl pH 8.5 (Teknova, T5085), 0.5 U/μL RNase inhibitor (Clontech, 2313B), 8 mM dithiothreitol (DTT) (Promega, P1171), 30 mM NaCl (Thermo Fisher Scientific, AM9760G), 2.5 mM MgCl2¬ (Thermo Fisher Scientific, AM9530G), 1 mM GTP (ThermoFisher Scientific, R0461), 2 μM TSO (Integrated DNA Technologies, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′), 5% polyethylene glycol (Sigma, P1458), and 2 U/μL Maxima H-minus reverse transcriptase (ThermoFisher Scientific, EP0753) in UltraPure water). Plates were thoroughly agitated to ensure mixture, spun down, then incubated in a C1000 Touch Thermal Cycler (BioRad) with the following program: (1) 42°C for 90 min for RT reaction, then (2) 70°C for 15 min to terminate the reaction.  For preamplification, 7.5 uL of PCR mix was added to each well (1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and 0.17 μM IS PCR primer (Integrated DNA Technologies, 5′-AAGCAGTGGTATCAACGCAGAGT-3′) in UltraPure water). Plates were again thoroughly agitated, spun down, then incubated using the following program: (1) 98°C for 3 min, (2) 98°C for 20 sec for denaturing, (3) 67°C for 15 sec for annealing, (4) 72°C for 6 minutes for elongation, (5) repeat from step 2 24 times, and (6) 72°C for 5 minutes. Preamplified cDNA was purified to remove small oligos and residual reaction components. cDNA purification was performed by adding 0.65X volume of calibrated AMPure XP beads (Beckman Coulter, A63882) to each well, followed by two washes with 80% EtOH, and finally elution in 12.5 μL UltraPure water.  Quality control Quality control was performed on the purified cDNA of each single cell on Fragment Analyzer (Advanced Analytical), a capillary electrophoresis-based machine. From the purified cDNA, 1 μL was taken from each single cell for analysis of concentration and size distribution. We set a minimum cDNA concentration cutoff of 1.7 ng/μL based on the cDNA concentration of empty wells containing ERCC but no sorted cell. Cells meeting the cDNA concentration cutoff were consolidated from 96 well plates into 384 well plates using a Mosquito X1 liquid handler (SPT Labtech). During consolidation, the cDNA concentration of each single cell was normalized to be within the 1.7–4.0 ng/μL range by diluting with an appropriate amount of UltraPure water.  Library preparation and sequencing Library preparation steps were carried out in 384-well plate format using a Mosquito HTS liquid handler (SPT Labtech). Tagmentation was performed by combining 0.4 μL cDNA from each single cell with 1.2 μL Tn5 mix (1 ng/μL homebrewed Tn5 enzyme, 16 mM Tris-HCl pH 7.6, 16 mM MgCl2 (Thermo Fisher Scientific, AM9530G) and 8% dimethylformamide (DMF) (Thermo Fisher Scientific, AC327171000) in UltraPure water. The tagmentation reaction was incubated at 55°C for 10 min. The reaction was terminated by adding 0.4 μL neutralization buffer (0.1% SDS). Indexing PCR reactions were performed using a custom made 7680-plex unique dual-index primer set (Integrated DNA Technologies). 0.4 μL of 5 μM i5 indexing primer, 0.4 μL of 5 μM i7 indexing primer, and 1.2 μL KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) was added to each well. Indexing PCR amplification was performed with the following program: (1) 72°C for 3 min, (2) 95°C for 30 sec, (3) 98°C for 10 sec for denaturing, (4) 67°C for 30 sec for annealing, (5) 72°C for 60 sec for elongation, (6) repeat from step 3 10 times. Following this amplification, 1 μL reaction product was taken from each well and pooled, forming a 384-cell pool. Each 384-cell library pool was purified using 0.8X volume of AMPure XP beads (Beckman Coulter, A63882), then analyzed on Fragment Analyzer (Advanced Analytical) for concentration and size distribution. Up to 20 384-cell library pools were normalized for concentration, pooled into a 7680-cell library pool, and purified again using 0.8X AMPure beads. The final 7680-cell library pool was sequenced on a NovaSeq 6000 S4 flow cell (Illumina) for 1-2 million 2×150 bp paired-end reads per cell.