GSM7776348: HL-1-SMC3-KD-rep2; Mus musculus; RNA-Seq
For 3C-HTGTS, mouse thymus and liver were removed, cells were filtered through nylon mech.10 million cells were cross-linked with 1% formaldehyde. Cell were lysed and digest with 200U MboI overnight at 37°C. The sample was ligated with T4 DNA ligase overnight at 16°C. Crosslinks were reversed by Proteinase K and RNase A prior to DNA extraction with 1:1 phenol-chloroform and precipitation with ethanol. The 10ug 3C DNA was sonicated to 300-500bp and was linearly amplified with a biotinylated primer. The biotin-labeled single-stranded DNA products were enriched with streptavidin C1 beads, and followed by 3' ends ligation with the bridge adaptor. The adaptor-ligated products were amplified via nested PCR using a nested primer and an adaptor-complementary primer. And a final PCR for another 5-10 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp or 250 bp paired-end reads. For SnRNA-seq, the E12.5 mouse embryos were microdissected in PBS solution using a Leica M205C microscope, and the heart tissues were extracted. The whole heart samples from different embryos, including six Smc3fl/fl, seven Smc3fl/+; Nkx2.5-Cre, and seven Smc3fl/fl; Nkx2.5-Cre, subjected to mechanical trituration, were made into the cell suspension. Cell counts and viability were determined for the resulting suspension, and the cell concentration was adjusted to the ideal 300-600 cells / μL. The prepared single-cell suspension was combined with a mixture of gel beads containing barcode information and enzymes and then encapsulated by oil droplets to form Gel Bead-In-EMulsions (GEMs). After the reverse transcription reaction, the GEMs were fragmented to complete the amplification of nuclear cDNA, and the quality-checked amplification products were constructed as sequencing libraries. Briefly, the cDNA was broken into fragments of 200-300 bp, end-repaired, ligated into the P7 Adapter, and finally amplified by PCR for introduction into the indexes of samples.