| Growth Protocol: |
Egg lays occurred in 2 hour intervals after 3x 1 hour pre-lays; embryos were aged for 6 hours (D. melanogaster) at room temperature.; Cells were grown in DMEM (Invitrogen 61965-026) without antibiotics containing 10% fetal bovine serum, and confirmed to be mycoplasma-free (LookOut Mycoplasma PCR detection kit, Sigma Aldrich). |
| Treatment Protocol: |
fixation: 80% methanol/PBS; Cells were grown to 30 to 60% confluence, dissociated with 0.05% bovine trypsin-EDTA (Invitrogen 25300062), quenched with growth medium, and further processed as described previously (Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1). condition: live; Cells were grown to 30 to 60% confluence, dissociated with 0.05% bovine trypsin-EDTA (Invitrogen 25300062), quenched with growth medium, and further processed as described previously (Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1). condition: fixed; Cells were grown to 30 to 60% confluence, dissociated with 0.05% bovine trypsin-EDTA (Invitrogen 25300062), quenched with growth medium, and further processed as described previously (Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1). condition: fixed, stored for 1 wk; Cells were grown to 30 to 60% confluence, dissociated with 0.05% bovine trypsin-EDTA (Invitrogen 25300062), quenched with growth medium, and further processed as described previously (Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1). condition: fixed, stored for 3 wk |
| Extract Protocol: |
Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2019 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2020 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2021 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2022 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2023 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2024 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2025 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension.Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1.; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2026 Ce; Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension. Macosko et al. 2027 Ce |
| Library Construction Protocol: |
We evaluated and quantified 1 μl of the amplified cDNA libraries on a BioAnalyzer High Sensitivity Chip (Agilent, Santa Clara, CA, USA). Then 600 pg of each cDNA library was fragmented and amplified (12 cycles) for sequencing with the Nextera XT v2 DNA Library Preparation kit (Illumina, San Diego, CA, USA) using custom primers that amplified only the 3' ends [7]. Libraries were purified with a 0.6× volume of AMPure XP beads followed by a 0.6–1× volume of AMPure beads to completely remove primer dimers and achieve an average length of ~500–700 bp, quantified and sequenced (paired end) on Illumina NextSeq 500 sequencers (library concentration: 1.8 pM; 1% PhiX spike-in for run quality control; NextSeq 500/550 High Output v2 kit (75 cycles); read 1: 20 bp (bases 1–12 cell barcode, bases 13–20 UMI; custom primer 1 Read1CustSeqB), index read: 8 bp, read 2 (paired end): 64 bp).; - |
