GSM8832718: NeMECiS_D6neural_progenitors_0nMSAG_Rep2; Mus musculus; OTHER
Plasmid library: 200mL of the OD600=1.0 bacterial culture was extracted using the Qiagen Plasmid Plus Maxi Kit (12963), according to manufacturer's instructions. Neural progenitors: To extract genomic DNA, sorted cells for the NeMECiS screen were pelleted in 1.5mL DNA LoBind microfuge tubes (Eppendorf, Cat No. Z666548) by centrifugation for 10 minutes at 500g. Genomic DNA was extracted for sorted and unsorted pellets using a Quick-DNA Miniprep Plus Kit according to the manufacturer's instructions (Zymo Research, Cat No. D4069), dividing each sorted sample into six columns each. Each column was eluted in 75 L of the supplied elution buffer (10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA) pre-warmed to 60ºC, into 1.5mL DNA LoBind microfuge tubes, pooling the samples of the same origin to bring the total eluted gDNA to 450uL. Barcode concatemers were amplified by PCR using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, Cat. No M0541L) by combining on ice: 450 L gDNA, 500 L 2X master mix, 5 L each of 100 M Nextera-adaptor primers (indexing each sample, see below), and 40 L nuclease-free water. Amplification was carried out under the following program: 5 minutes at 72°C, 30 seconds at 98°C, followed by 17 cycles of 30 seconds at 98°C, 30 seconds at 63 °C and 60 seconds at 72°C, and subsequently 5 minutes at 72°C. At this step, all PCR products were pooled. The pooled amplicon DNA was then cleaned up using 1.8x v/v of AMPureXP beads (Beckman Coulter, Cat. No. A63882) and eluted in 50 L EB buffer (10 mM Tris-Cl, pH 8.5). Library concentration was determined using the KAPA Library Quanitification Complete Kit Universal (Roche, Cat No. 07960140001) according to manufacturer's instructions, using the QuantStudio 12K Flex Real-Time PCR System for qPCR (Applied Biosystems, Cat No. 4471087). 50% of the amplified sequence was further amplified using NEBNext High-Fidelity 2X PCR Master Mix and P5 (AATGATACGGCGACCACCGA) and P7 primers (CAAGCAGAAGACGGCATACGAGAT) using the same programme as above, then re-quantified as above to establish a concentration in excess of 1nM. Size spectra were examined with a Bioanalyzer using the DNA High Sensitivity DNA Kit (Agilent, Part No. 5067-4626) to confirm library quality. The plasmid library was sequenced using the Illumina MiniSeq Sequencer on a 'High Output' run using the MiniSeq High Output Reagent Kit (300-cycles) (Illumina, Cat No. FC-420-1003) according to the manufacturer's instructions. The neural progenitor library was sequenced using the Illumina NextSeq 500 Sequencer on a 'High Output' run using the NextSeq 500/550 High Output Kit v2.5 (300 Cycles) (Illumina, Cat No. 20024908) according to the manufacturer's instructions.  Sequence demultiplexing was performed with the built-in software by supplying the corresponding indexing sequences for each sample.