GSM6398106: sci-RNA-seq3 with ETiX mouse embryoids and natural mouse embryos [NextSeq2_PA-06D]; Mus musculus; RNA-Seq
We performed a simplified version of sci-RNA-seq3, further optimized for “tiny” samples (Martin et al. 2021). Briefly, to each tube, 100ul of a hypotonic, PBS-based lysis buffer was added with DEPC as an RNase inhibitor. The resulting nuclei were then fixed with 4 volumes of a mix of methanol and dithiobis (succinimidyl propionate) (DSP). After rehydrating and washing the nuclei carefully in a sucrose/PBS/triton/MgCl2 buffer (SPBSTM), the nuclei were distributed to two 96-well plates for reverse transcription, allocating 8 wells per embryo. After reverse transcription, nuclei were pooled, washed in SPBSTM and redistributed to a fresh plate for ligation of the second index primer with T4 DNA ligase. Nuclei were then again pooled, washed, and redistributed to three final plates for second strand synthesis, extraction, tagmentation, and PCR to add the third index plus a plate index. Products were pooled by PCR plate, size-selected and sequenced on two Illumina NextSeq runs (termed “NextSeq-1" and “NextSeq-2”).