Sera mag oligo dt beads

Library preparation and high throughput sequencing were performed at Beijing Ori-Gene Science and Technology Ltd. Total RNA were prepared to isolate mRNA. Using Sera-mag Oligo (dT) beads (Thermo), 10 μg of total Poly(A) mRNA was isolated, which was cut into short fragments to establish a paired-end RNA-seq library for transcriptome sequencing. Adapters were added for size selection and PCR amplification. Fragment shortening was interrupted by addition of a stop buffer. using these fragments as templates, the first-strand cDNA was synthesized with a random hexamer-primer. 20 μL second-strand buffer (Invitrogen), 10 mM dNTP Mix, 5 U/μL RNase H, and 10 U/μL DNA polymerase I were used to produce the second-strand cDNA. Using the QIAquick PCR purification kit short fragments were purified, and then resolved in EB buffer for end reparation and poly(A) addition, which was followed by connection with sequencing adapters. Thereafter, suitable fragments were used as templates to be amplified by PCR. Finally, according to Illumina’s protocols, the paired end RNA-seq libraries of the samples were prepared. Then, the constructed libraries were sequenced by the Illumina HiSeq. 2500 platform.

Mixed-stage embryos were collected from N2, TY1916, BC4289 and SP117 by bleaching gravid adults. L2-L3 worms were collected by plating embryos and growing for 22–26 h at 22.5°C, and young adults next day. Worms were resuspended in at least 10 volumes of Trizol and stored at −80°C. RNA preparation was performed as previously (Albritton et al. 2014 (link)). Briefly, samples were freeze cracked 3–5 times, followed by TRIzol purification, and cleaned up with Qiagen RNeasy kit. From 0.5 to 10 μg of total RNA, mRNA was purified using Sera-Mag oligo(dT) beads (Thermo Scientific), sheared and stranded Illumina sequencing libraries were prepared using a previously published protocol (Parkhomchuk et al. 2009 (link)). Sequencing was performed with Illumina HiSeq-2000 and reads were aligned to genome version WS220 with Hisat2 version 2.2.1 (Kim et al. 2019 (link)) using default parameters. Count data were calculated using HTSeq version 0.13.5 (Putri et al. 2022 (link)) and differential expression was performed using the R package DESeq2 version 1.30.1 (Love et al. 2014 (link)). FPKM values were generated using Cufflinks version 2.2.1 with options -p 8 –library type fr- first strand (Trapnell et al. 2010 (link)). FPKM and DEseq2 output are provided in Supplementary File 4. In Fig. 3, log2 fold change values were median centered by subtracting the genome median from each value.

Ebf2-EGFP positive and negative cells were dissociated from the cerebral cortex and collected by FACS on E11.5, E13.5 and E15.5 stages, respectively. Approximately 12 Ebf2-EGFP positive embryos from three mice were used in each experiment at each stage and 10 million cells for each sample were collected with two biological replicates. Total RNA was extracted using QIAGEN RNeasy Mini Kits and processed by Sera-Mag Oligo (dT) beads (Thermo Scientific), libraries were prepared using the NEB Next mRNA Sample PreP Master Mix Set 1. All these experiments were performed according to the manufacturer’s instruction. Samples were sequenced on Illumina HiSeq 2000 using 100 base paired-end reads.