GSM8286378: Mouse embryo replicate 2; Mus musculus; RNA-Seq
Hela cells were cultured at 37 °C in a humidified 5 % CO2 atmosphere, in high-glucose DMEM (Gibco; 11965092) supplemented with 10 % calf serum (Gibco;10270-106), 1 % penicillin/streptomycin (Nacalai Tesque; 0503), and 1 % L-glutamine (Nacalai Tesque; 16948-04). For cell preparation, Poly-l-lysine-coated chambers were coated with sterile PDL (Sigma Aldrich cat. no. P6407) at 0.3 mg/ml overnight at 4 °C. Afterwards, the chamber was dried for 1 hour, washed with UltraPure water (Invitrogen; 10977023). Chambers were then seeded with cells per well and placed in an incubator (37 °C with 5 % CO2) two days for subcellar resolution validation.Fresh frozen tissue (moouse embryo E14.5 and mouse brain) were OCT embedded, and 10um fozen sections were cut with cryostat. FFPE tissue (mouse embryo E15.5) is sliced with 7um. Different barcode represent different region of light illumination. The library struction is "read2-7bp bar—32fix--8bp-UMI—22bp dTVN-cDNA-read1" The library was performed by IVT and RT-PCR protocol. Briefly,The RT products (15 μl) were combined with PCR mix, and amplified by PCR . Fragments of 250–1000 bp were then purified and size-selected with Speedbead magnetic capboxylate. The quality of the resulting cDNA library was quantified using a Qubit dsDNA HS assay kit (Invitrogen, Q32854) with Qubit 3.0 Fluorometer (Invitrogen),before sequencing on the Illumina NovaSeq 6000.