title: GSM4013404: Day 120 aging - worker - rep1; Harpegnathos saltator; RNA-Seq
library_construction_protocol: Brains were removed from ants and optic lobes were removed in DMEM medium, then washed twice with DMEM medium. 1 mL of papain solution (5 mL DMEM medium/vial with 45 uM actinomycin D) was added and brains were rotated at room temperature for 12 minutes. Following two washes with DMEM medium and one wash with PBS solution (1x PBS with 0.01% BSA), brains were resuspended in 200 µL PBS solution, pipetting up and down. Samples were incubated for 5 minutes and pipetting was repeated. Samples were filtered with a 40 µm strainer to obtain a single-cell suspension. For 10x sequencing, cells were loaded to RT mix to aim for sampling of 5,000–10,000 cells. All downstream cDNA synthesis (12 PCR cycles), library preparation, and sequencing were carried out as instructed by the manufacturer (10x Genomics Chromium Single Cell 3' Reagent Kit v2 User Guide RevD) with minor modifications; custom barcodes were used as indexes, libraries were loaded at 2.0 pM, and the read configuration was 26 bp (read 1), 8 bp (index 1), and 58 bp (read 2). For Drop-seq, the final cell suspension was diluted to 100 cells/µL with PBS solution and 1.5 mL of the cell suspension was loaded for each Drop-seq run. Barcoded beads were resuspended in freshly made lysis buffer, composed of 200 mM Tris-HCl (pH 8.0 at room temperature), 20 mM EDTA, 6% Ficoll PM-400 (GE Healthcare/Fisher Scientific), 0.2% sarkosyl (Sigma Aldrich), and 50 mM DTT, at a concentration of 120 beads/mL. The flow rate for cells and beads were set to 4,000 mL/h, while droplet generation oil (Bio-rad) was run at 15,000 mL/h. Droplets were generated and collected in a 50 mL Falcon tube for a run time of 15 minutes. Droplet breakage with Perfluoro-1-octanol (Sigma-Aldrich), reverse transcription, exonuclease I treatment, and amplification of cDNA were performed following the Drop-seq protocol of the McCarroll lab (http://mccarrolllab.org/dropseq/), with minor modifications. cDNA was amplified using Terra PCR Direct Polymerase Mix Kit. After two rounds of purification with 0.6x SPRISelect beads, we tagmented 600 pg DNA using the Nextera XT DNA Sample Preparation Kit (Illumina, cat # FC-131-1096). Libraries were further amplified with 12 PCR cycles using custom P5-TSO hybrid and custom Nextera-compatible primers with different indexes. Libraries were loaded at 2.0 pM. Custom read 1 and index 2 primers were used, and libraries were sequenced on an Illumina NextSeq500 using a 75-cycle High Output v2 Kit (Illumina, cat # FC-404-2005). The read configuration was 20 bp (read 1), 8 bp (index 1), 8bp (index 2), and 56 bp (read 2).