GSM6235598: Tet1/2 DKO chimera plate 4; Mus musculus; RNA-Seq
Whole-embryo chimera, mixed chimera, and AAV-Cre treated embryos were dissected in ice-cold 1×PBS, followed by imaging in a drop of DMEM medium supplemented with 10% foetal bovine serum (FBS) under a Nikon Eclipse Ti2 inverted microscope. Embryos were then rinsed in ice-cold 1×PBS, transferred into an Eppendorf tube containing 35 µl 0.25% Trypsin/0.02% EDTA Solution A, and incubated at 37 °C for 5 min with 2-3 times pipetting. Trypsinization was stopped by adding 10% FBS/DMEM medium. Cell suspensions were filtered through a 35 µm strainer before sorting. For MARS-seq analysis, single cells were index-sorted into a pre-barcoded 384-well capture plate that contains 2 µl of lysis solution and barcoded poly (T) reverse-transcription (RT) primers. In each plate, 12 wells at positions C1, C2, D1, D2, J6, J7, K6, K7, O23, O24, P23, and P24 were kept as non-cell controls. Immediately after sorting, each plate was sealed, spun down, snap-frozen on dry ice, and stored at -80 °C until library preparation. For bisulfite sequencing, Tet-TKO1, Tet-TKO2, Ctrl1, and Ctrl2 cells were sorted from E8.5 chimera embryos respectively. Cell pellets were kept at -80 °C until library preparation. Genomic DNA was extract using commercial kit. Single-cell cDNA libraries were prepared using the MARS-Seq method as described (Keren-Shaul et al., 2019). Briefly, reverse transcription was performed in individual wells of the sorted 384-well plate to get complementary DNA (cDNA). Subsequently, all liquid from each 384-well plate was pooled and converted to double-stranded DNA, transcribed using T7 RNA polymerase, and fragmented. A unique plate adaptor was added to each library to be sequenced together, and RNA::ssDNA (plate adaptor) ligation was performed. The purified ligates were applied to a second RT step with a primer complementary to the ligated adaptor, and the library is completed by cDNA enrichment through PCR amplification. Library concentration and profile were evaluated before sequencing.  Bulk PBAT library was performed as previously described (Meir et al., 2020). Briefly, genomic DNA was converted with bisulfate, and applied to the first and second strand synthesis. The product was treated by Klenow fragment and purified. A final PCR was performed using a universal primer and indexed primers. The final product was clean and sequenced.