title: GSM8212853: neural progenitors, mScarlet, rep 2; Mus musculus; ATAC-seq
library_construction_protocol: On day 9, cells were sorted for mScarlet3 fluorescence and 50,000 per sample were used for ATAC-seq library preparation. Samples were resuspended in 0.5 ml of RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% Igepal CA-630 (Sigma, Cat. No. I8896-100ML), 0.1% Tween-20 (Sigma, Cat. No. P2287-500ML), and 0.01% digitonin (Invitrogen, Cat No. BN2006), and incubated on ice for 3 min. 1 ml of RSB with 0.1% Tween-20 was then added and samples were spun for 10 min at 2000 g and 4ºC. The samples were then resuspended in 1 ml of ATAC mix (2X TDE buffer , 50 µl Tn5 enzyme, 0.01% digitonin, 0.1% Tween-20 and 330 µl of PBS). Transposition was carried out at 37ºC with 1000 rpm shaking for exactly 30 min. DNA was isolated using the DNA clean up & Concentrator kit (Zymo, Cat. No. D4013) as per manufacturer's instructions. DNA was eluted in 21 µl of DNAse-free water. DNA was stored at -20ºC until ready to be processed. Transposed DNA was first amplified with 2X PCR Master Mix NEB (Cat. No. M0541S), and 1 µl of constant forward primer and indexed reversed primer (Table S1) (both at 25 µM) in a total reaction volume of 50 ul. The program was as follows: 5 min at 72ºC, 30 s at 98ºC, followed by 7 cycles of 30 s at 98ºC, 30 s at 63 ºC and 60 s at 72 ºC, with a final extension of 5 min at 72 ºC. DNA was cleaned up with 1.8x volumes of AMPureXP beads (Beckman Coulter, Cat. No. A63882) and eluted in 18 µl of EB buffer. qPCR was carried out to determine the number of extra cycles using 2 µl of the amplified DNA in technical duplicates. Each 20 µl reaction contained 20 µl of 2X SYBR Green PCR assay, using PowerUP SYBR Green Master Mix (Thermo Fisher, Cat. No. A25742) and 2.5 µl of each primer (at 25 µM each). The qPCR program was as follows: initial activation for 2 min at 50ºC, 30 s at 98ºC, followed by 40 cycles of 10 s at 98ºC, 30 s at 63ºC and 60 s at 72ºC. The number of additional cycles was calculated as ¼ of the maximum amplification 86. The second amplification was done using 12.5 µl of amplified DNA, 2X PCR Master Mix NEB and 2.5 µl of each primer (at 25 uM) in a total volume of 50 ul. The program was the same as the first amplification without the initial extension and for the calculated number of cycles. Finally, re-amplified DNA was cleaned up using 1.8x volumes AMPureXP beads and eluted in 30 ul.