title: GSM5969110: MM-expr02-plate01-082; Mus musculus; RNA-Seq
library_construction_protocol: The snap frozen embryos were processed as previously described (Cao et al. 2019). Briefly, the frozen embryos were cut into small pieces with a blade and further dissected by resuspension in 1 ml ice cold cell lysis buffer (CLB) in a 6 cm dish. adding another 3ml CLB, the sample was strained (40 µm) into a 15 ml Falcon tube and centrifuged to a pellet (500g, 5 min). Resuspending the sample with another 1 ml CLB, the isolation of nuclei was ensured. Pelleting the isolated nuclei again (500g, 5 min) was followed by a washing step by fixation in 10 ml 4% Paraformaldehyde (PFA) for 15 minutes on ice. The fixed nuclei were pelleted (500g, 3 min) and washed twice in the nuclei suspension buffer (NSB) (500g, 5 min). The nuclei finally were resuspended in 500µl NSB and split into 2 tubes, each containing 250 µl sample. The tubes were flash frozen in liquid nitrogen, until further use for library preparation. The embryo preparation was preceded randomly for nuclei isolation in order to avoid batch effects. The library preparation proceeded as previously described (Cao et al. 2019). In short, the fixed nuclei were permeabilized, sonicated and washed. Nuclei from each mouse embryo were then distributed into several individual wells into 4 96-well plates. We split samples into four batches (~25 samples randomly selected in each batch) for sci-RNA-seq3 processing. The ID of the reverse transcription well was linked to the respective embryo for downstream analysis. In a first step the nuclei were then mixed with oligo-dT primers and dNTP mix, denatured and placed on ice, afterwards they were proceeded for reverse transcription including a grading incubation step. After reverse transcription, the Nuclei from all wells were pooled with the nuclei dilution buffer, spun down and redistributed into 96-well plates containing the reaction mix for ligation. The ligation was proceeded for 3 h at 16 °C. Afterwards, nuclei again were pooled with nuclei suspension buffer, spun down and washed and filtered. Next, the nuclei were counted and recistributed for second strand synthesis, which was was carried out at 16 °C for 3h. Afterwards tagmentation mix was added to each well and  tagmentation was carried out for 5 minutes. Stopping the reaction, DNA binding buffer was added and the sample was incubated for another 5 minutes. Following an elution step  using AMPure XP beads and elution mix, the samples were PCR amplified. Finally after PCR amplification, the nuclei were pooled and purified using AMPure XP beads. The library was analyzed by electrophoresis and the concentration was calculated using Qubit (Invitrogen). The library was sequenced on the NovaSeq platform (Illumina) (read 1: 34 cycles, read 2: 52 cycles, index 1: 10 cycles, index 2: 10 cycles).