GSM5460934: Pectoral and pelvic fin motor neurons - ATAC-seq rep-1; Leucoraja erinacea; ATAC-seq
In stage 30 little skates, pectoral and pelvic motor neurons were labeled by injecting rhodamine-dextran (3K, Invitrogen). After 1-2 day incubation at RT, cell dissociation was performed. Spinal cords were isolated and digested with pronase for 30min at RT and dissociated motor neurons from several labeled spinal cords were collected manually. 150-426 labeled motor neurons were collected in 25µl cold ATAC-seq RSB (10mM Tris-HCl pH7.4, 10mM NaCl, and 3mM MgCl2 in Nuclease free water) and centrifuged at 500rcf for 10min in a pre-chilled fixed-angle centrifuge. The supernatant was removed and 10µl of transposition mix (3.3µl PBS, 1.15 µl Nuclease free water, 5 µl 2X TD buffer, 0.25 µl (Tn5, 1/10 diluted: 5µl 2X TD buffer, 4µl nuclease free water, 1µl Tn5), 0.1 µl 1% digitonin) was added and resuspended by pipetting six times. The samples were incubated with shaking at 1000rpm for 30min at 37℃. Tagmented DNA was purified with Qiagen minelute reaction cleanup kit (Qiagen, 28206). PCR mixtures (10µl Nuclease free water, 2.5µl 25µM primer (Ad1), 2.5µl 25µM primer (Ad2.X), 25µl 2X NEB master mix, Transposed sample 20µl) were prepared and primary PCR was performed in the following condition: 72℃ 5min, 1cycle; 98℃ 30sec, 1cycle; 98℃ 10sec, 63℃ 30sec, 72℃ 1min 5cycles; hold at 4℃. PCR mixture (3.76µl nuclease free water, 0.5µl 25µM primers (Ad1.1), 0.5µl 25µM primers (Ad2.X), 0.24µl 25X SybrGold in DMSO, 5µl 2X NEB master mix, 5µl pre-Amplified sample) were prepared and quantitative PCR was performed as follows: 98℃ 30sec, 1cycle; 98℃ 10sec, 63℃ 30sec, 72℃ 1min 20cycles; hold at 4℃. 1/4 max cycles were determined (N) and the primary PCR reaction products underwent N more PCR cycles. For the tail region spinal cord, 240-1000 cells from two embryos were used for ATAC sequencing library preparation.   The primes used in the ATAC library preparation were as follows:  AD1.1:AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTG; AD2.1:TAAGGCGACAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT; AD2.2:CGTACTAGCAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGT; AD2.3:AGGCAGAACAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT; AD2.4:TCCTGAGCCAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT; AD2.5:GGACTCCTCAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGT