NextSeq 500 paired end sequencing; Single-cell chromatin accessibility profiling of mesoderm development in wild-type and mutant embryos
Single-cell chromatin accessibility profiling of mesoderm development in wild-type and mutant embryos
Drosophila melanogaster embryos were collected and fixed as previously described (Sandmann et al., 2007). In summary, embryos were collected in staged two-hour windows following three one hour pre-lays to clear the females and synchronize the collections, which were aged at 25C to the corresponding time window (3-5 hr, 4-6 hr, 5-7 hr, 6-8 hr, 7-9 hr, 8-10 hr, 9-11 hr and 10-12 hr for wild-type collections, 10-12 hr for Mef2, 5-7 hr for tinman and 6-8 hr for biniou and bagpipe mutant embryo collections). Embryos were dechorionated in 50% bleach for 2 min and fixed with 1.8% formaldehyde for 15 min. For Mef2 hand-sorted mutant embryo collections, homozygous mutant embryos (GFP negative) were hand-sorted from their GFP marker balancer siblings using the stock dMef2[22.21]/CyO, twi-Gal4, UAS-GFP, under a dissection microscope prior to fixation. After 15 minutes of formaldehyde fixation, embryos were quenched with glycine, washed, dried, snap frozen in liquid nitrogen and stored at _ 80C. Frozen embryos were transferred into a glass douncer placed on ice and thawed in HB buffer with proteinase inhibitor. Embryos were dounced 20 times with the loose pestle and 10 times with the tight pestle. The homogenate was filtered through two layers of Miracloth, centrifuged and the supernatant was discarded. The nuclei pellet was washed once in HB buffer and resuspended in PBT. The nuclei were dissociated by passing them 10 times through a 20G needle and 10 times through a 22G needle using a 5 mL syringe and then filtered through 20 uem Nitex membrane. The extracted nuclei were quantified using the LSRFortessa system, snap frozen in liquid nitrogen and stored at -80C. Generation of sci-ATAC-seq libraries was performed largely as previously described (Cusanovich et al., 2018), with some modifications. Nuclei were washed twice by pelleting and resuspending in 1 mL PBS supplemented with 0.1% TritonX-100 and 1_ protease inhibitor cocktail (Roche). Nuclei were stained with 3 _M DAPI and 2,500 DAPI+ (and Mef2+ or Biniou+ for mesoderm / visceral muscle sorting) nuclei were sorted into each well of a 96-well plate containing 5 _L of Omni-ATAC buffer (Corces et al., 2017) supplemented with 1_ protease inhibitor cocktail (Roche) and 12 _L of TD buffer (Illumina) in each well. Tagmentation was performed by adding 2 _l of each of the 96 custom and uniquely indexed Tn5 transposomes (Illumina, 2.5 uM) and by incubating at 55 C for 2 hours. Following the second sorting step and reverse-crosslinking, tagmented DNA was PCR amplified by adding 5 _L of forward and reverse indexed primers (Cusanovich et al., 2018a), 7.5 _l of NPM polymerase master mix (Illumina) and BSA (2X final concentration; NEB) to each well and by running the followingcycling conditions: 72 C 5 min, 98 C 30 s; 98 C 10 s, 63 C 30 s, 1920 cycles; 72 C 1 min, hold at 10 C. The optimal number of cycles for each library was determined beforehand by monitoring amplification on a qPCR machine for a set of test wells. Libraries were sequenced on an Illumina NextSeq 500 sequencer High Capacity 150 PE kit (Illumina) using custom primers and a custom sequencing recipe (Amini et al., 2014).