GSM9140740: L929 cells, 3' knock groups; Mus musculus; RNA-Seq
cells were detached with trypsin treatment after the supernatant was removed. Perform cell aggregate dissociation with strong pipetting. Cells were then rinsed three times with ice-cold phosphate buffer solution (PBS), pelleted by centrifugation at 500 ×g for 10 min at 4 °C to remove the supernatant containing excess trypsin and culture media thoroughly. The home-built microneedle capturing system described above was employed for picking up single cells into 8- or 96-well plates containing lysis buffer. Specifically, under the optimized diameter of the capillary tip (30 μm), the conical tip of the capillary probe was controlled to be aligned with the target cells, and the target cell was sucked into the probe under capillary force. The CBTi-seq lysis buffer consisted of 0.13 µl recombinant RNase inhibitor (40 U/µl, Takara), 0.25 µl of Takara lysis buffer (10 ×) and 0.5 µl CBTi-Oligo dT primer (10 µM), and ddH2O to 2.5 µl final volume. The plates were centrifuged at 1000 ×g immediately after sorting and stored at −80 °C until use. Sorted single-cell or microregion tissue samples were removed from the −80 °C storage and thawed at room temperature for 10 min to facilitate sample lysis and RNA release, then transferred to a preheated PCR thermocycler, incubated for 3 min at 72 °C and immediately placed on ice afterwards. Next, 10 µl of RT-PCR mix containing 1.2 µL dNTP mix (10 mM), 0.252 µL RNase Inhibitor (40 U/µl), 0.1 µL DTT (100 mM), 0.328 µL MgCl2 (279 mM), 0.18 µL dCTP (100 mM), 2 µL Betaine (5 M), 0.368 µL CBTi-TSO (50 µM), 0.172 µL of Superscript Ⅳ / Maxima H (200 U/µL, ThermoFisher Scientific) and 5 µL of KAPA HiFi HotStart Ready Mix (2×, Roche) were added to synthesis full-length dsDNA. The following RT-PCR program was set to: 60 min at 50 °C, 98 °C for 3 min, then 20 cycles of (98 °C for 20 s, 67 °C for 20 s, 72 °C for 6 min). The cDNA concentration was quantified using the Qubit 4.0 fluorimeter, and size distribution was assessed using capillary electrophoresis on a Fragment Analyzer system (Agilent Bioanalyzer). 1 μl unpurified amplified cDNA working dilution (5 ng/µl) of each sample was transferred to a new plate before adding tagmentation reaction mix as described above. Of note, each sample used unique combination barcoding. Samples were incubated for 10 min at 55 °C. Then, all tagmented products were mixed into 1.5- or 2 ml PCR tube, purified with 1.5× AMPure XP beads for termination of the tagmented reaction and collection, followed by elution with 20 µl nuclease-free water.