GSM9651340: Parse sublibrary 2; Mus musculus; RNA-Seq
Plugged C57BL/6J females were purchased from Janvier Labs and E0.5 was defined as the morning on which the vaginal plug appeared. Mice were subjected to in utero electroporation and embryos harvested as previously described41. Electroporation was performed using the pCAG-mScarlet-3XFlag vector encoding for Nfib-WT, Nfib-S265A, Hmgn3-WT, Hmgn3-S78A, and an empty vector served as a control. In brief, plasmids were prepped using an NncleoBond Xtra Maxi Endotoxin-free Plasmid maxi kit (Cat no: 740424.10) and plasmid concentration adjusted to 1.5 μg/μl. Pregnant mice were anaesthetised with isofluorane at E13.5 and ∼1.5 μl of plasmid solution was injected into the ventricle of embryonic brains. The plasmids were electroporated into the dorsal ventricular zone with six pulses of 30 V, 50 ms each at 1 s interval delivered through platinum electrodes using round electrodes CUY650P5 and Nepa Gene electroporator (NEPA21 Type II). After four days of electrophoration (E17.5), the brain tissues were processed for the analyses. For the isolation of electroporated cells from E17.5, the dorsolateral neocortex was dissected, and a single cell suspension was produced from two to three hemispheres per condition using the MACS Neural Tissue Dissociation kit containing papain (Miltenyi Biotec, 130-092-628). The cells were resuspended to 1 million cells/ml in FACS buffer (3%BSA, 1mM EDTA in HBSS w/o Ca2+ and Mg2+), passed through a 40-μm filter and immediately processed by FACS on a five-laser Invitrogen BIGFOOT cell sorter. For each condition, 30,000 to 100,000 cells were collected in 1.5 mL DNA LoBind tubes containing FACS buffer. scRNA-seq libraries were generated according to Parse Biosciences protocols: Evercode Cell Fixation v3 and Evercode WT Mini v3. For cell fixation, FACS sorted single cell suspensions were centrifuged for 10 min at 200g at 4°C in 15-ml BSA-coated Falcon tubes and resuspended in Cell Prefixation Buffer with BSA 7.5% (Gibco) in 15-ml BSA-coated Falcon tubes. Cell suspensions were incubated in fixation solution followed by the addition of permeabilization solution and stop buffer, according to the manufacturer's instructions. Cell suspensions were frozen using a Mr. Frosty Freezing container (Sigma-Aldrich) at −80°C until further use. Samples barcoding and sub-libraries were prepared using the Evercode WT Mini v3 as per the manufacturer's instructions and sequenced on a NovaSeq 6000 instrument. Single-cell transcriptomic libraries were prepared using the Evercode WT Mini kit (Parse Biosciences, Chemistry v3) according to the manufacturer's protocol. Sequencing was performed on two sublibraries