GoKawiilTissue dissociation, sample fixation and storage
Cells were isolated from the developing human cortex between GW15 and GW24 using a method similar to that previously described in ref. 3. Dissociated cells were washed twice in PBS and fixed in 2% PFA for 10 min at room temperature. Fixation was quenched by adding glycine to a final concentration of 200 mM, followed by incubation for 5 min at room temperature. From this point onwards, all procedures were performed either on ice or at 4 °C. Fixed cells were pelleted by centrifugation at 1,000g, washed once with PBS, filtered through a 70-μm nylon mesh and washed once again with PBS. Finally, the cells were pelleted and stored at −80 °C.
FACS
All procedures were performed either on ice or at 4 °C. About 1.5 × 108 fixed cells were thawed and permeabilized by incubating in 1 ml of PBS containing 0.1% Triton X-100 for 15 min. Bovine serum albumin (BSA) was added to a final concentration of 1% and cells were pelleted by centrifugation at 1,000g for 8 min. Cells were washed once in staining buffers (PBS with 1% BSA) and resuspended in 100 µl of staining buffer. Cells were blocked by FcR Blocking Reagent (Miltenyi Biotech, 1:20) for 10 min, followed by antibody incubation for 30 min. The antibodies used for FACS included PerCP-Cy5.5 anti-SOX2 (BD Biosciences, 561506, for RG), PE-Cy7 anti-EOMES (Invitrogen, 25-4877-42, for RG), unconjugated anti-HOPX (Proteintech, 11419-1-AP, for RG), Alexa Fluor 647 anti-OLIG2 (Abcam, ab225100, for OPC/MG) and PE anti-PU.1 (Cell Signaling Technology, 81886, for OPC/MG). The HOPX antibody was used at 1:250 dilution, whereas all other antibodies were used at 1:20 dilution. After incubation, cells were washed twice in staining buffer, resuspended in 300 µl of staining buffer and incubated with the Alexa Fluor 647 donkey anti-rabbit secondary antibody (Invitrogen, A-31573, for RG FACS only) at 1:300 dilution. Cells were sorted using BD FACSAria II sorters into collection buffer (PBS with 5% BSA) (Supplementary Figs. 1 and 2). Sorted cells were pelleted by centrifugation at 1,000g for 10 min, snap-frozen on dry ice and stored at −80 °C before further processing. For FACS performed for RNA-seq, 1% RiboLock Rnase Inhibitor (Thermo Scientific, EO0384) was included in all buffers.
RNA-seq library creation and analysis
We extracted total RNA from the sorted cell populations using the RNA FFPE kit (Qiagen 73504) starting with 3 × 105 to 1.8 × 106 cells. The quality of the extracted RNA was checked by determining the percentage of RNA fragments with size larger than 200 bp (DV200) from the Agilent 2100 Bioanalyzer, with DV200 ≥ 30% used for library construction. Samples were then depleted of ribosomal RNA using the KAPA RNA HyperPrep Kit with RiboErase (HMR KK8560) and we performed first and second strand synthesis, dA-tailing and sequencing adapter ligation. Last, sequencing adapters were added by means of PCR amplification and libraries were sent for paired-end sequencing on the NovaSeq S4 instrument (100 bp paired-end reads).
Raw reads were trimmed to 100 bp using fastp75 (v.0.22.0) and then aligned to hg38 using STAR (v.2.7.10a) running the standard ENCODE parameters. Strand-specific quantification was performed using RSEM (v.1.2.28) with the GENCODE 38 annotation. Library quality was further evaluated with median TIN76 score and shown to be greater than 60 across all samples. TMM-normalized reads per kilobase per million mapped reads (RPKM) values for each gene were obtained by use of the edgeR77 (v.3.32.1) package. The mean values across all replicates were used for all downstream analyses. DEGs were identified with DESeq278 using a multi-factor design including the genotype or individual to identify differences between vRG and oRG. Clustering was performed on regularized log transformation data and hierarchical clustering based on sample distances after removing batch effects from genotype or individual with the removeBatchEffect command from limma79 (v.3.46.0).
Characterizing bulk RNA-seq with scRNA-seq
We leveraged CIBERSORTx80 to characterize cell composition with matching scRNA-seq9. eN, iN and IPC subclusters were first combined into one cluster before creating the reference matrix. Final counts consisted of eN, iN, IPC, vRG, oRG, truncated RG, OPC and MG cluster. Raw counts of bulk vRG, oRG, OPC and MG libraries from this study were obtained with tximport() from the rsem output. Finally, CIBERSORTx was used to impute cell fractions with the following parameters, batch correction B-mode, relative run-mode and 100 permutations.
ATAC-seq library creation and analysis
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