GoKawiilDestination vector cloning
We built a specific custom destination vector for efficient Golden Gate cloning of enhancer fragments. This included the reporter gene cassette NanoLuc-Ires-mNGreen-pA, downstream of a mCMV promoter. A bacterial suicide ccdB cassette spanning the enhancer position enabled assembly of a single enhancer for selection and efficient cloning. This was based on the EMMA Golden Gate cloning system43. For the GSC long-term differentiation experiments, the same vector was built but included PiggyBac transposase recognition sites flanking the entire cassette.
Bioinformatics to design a SOX2 enhancer oligonucleotide pool
We re-analysed previously published1 SOX2 ChIP–seq and H3K27ac GSC cell line data to identify GSC-specific SOX2 peaks that were overlapping with H3K27ac and absent in differentiated cells (cells in serum culture). The resulting shared peaks were then combined into one set and manually curated to remove centromeres. This resulted in 1,721 peaks with an average length of 402 bp. These peaks were then used to design a set of 160 bp sequences. Next, 20 bp adapters were included to flank the sequences and these sequences were synthesized as an oligonucleotide pool (Twist Bio).
Construction of an arrayed plasmid library of enhancer fragments
The oligonucleotide pool (9,523 unique sequences) was first amplified for 10 cycles with 0.25 µl (2.5 ng input) volume, and 0.5 µl of this reaction was used for the subsequent 15 cycles of amplification to reduce PCR ‘jackpot’ amplification. The final products were cloned into the expression vector using an efficient Golden Gate reaction. We used KAPA HiFi Hotstart polymerase with GC buffer (Roche, KK2501), 68 °C annealing temperature and 5 s of extension time at 72 °C. A total of 4,579 individual plasmids were then randomly picked, miniprepped and plated as an arrayed plasmid DNA library on 96-well plates. This was deemed a practical number for arrayed library screening. A magnetic-bead-based SPRI purification method was used to minimize loss and to clean up the DNA before Golden Gate cloning. Sanger sequencing for quality checks of a sample of the plasmids confirmed that they were diverse, and all sequences could be mapped back to the original library design.
Screening platform for 384-well plates
Cells were seeded in 384-well plates using a multidrop and transfected the following day using CyBio Felix (CyBio). Two days later, a NanoGlo DLR assay was used to identify hits with a fold change to mCMV of >10. These hits were Sanger sequenced to determine the specific sequence and mapped back to the genome to validate overlap with the original SOX2 ChIP–seq data. The GREAT tool predicted the target gene for hit sequences44.
General cell culture procedures
The GSC lines GCGR-E17 (E17), GCGR-E21 (E21), GCGR-E27 (E27), GCGR-E28 (E28), GCGR-E31 (E31), GCGR-E34 (E34), GCGR-E37 (E37) and GCGR-E55 (E55) and the human NSC lines NS9FB_B (NS9), NS12ST_A (NS12) and NS17ST_A (NS17) were generated in the Pollard Laboratory and are available upon request from the Glioma Cellular Genetics Resource. Informed consent was obtained for use of patient tissue. All procedures on patient brain tissue received ethics approval from the NHS Health Research Authority, East of Scotland Research Ethics Service (REC reference 15/ES/0094), and all procedures on embryonic and fetal brain tissue received ethics approval from the Lothian NHS Board, South East Scotland Research Ethics Committee (REC reference 08/S1101/1).
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