GSM7662984: Enteric glial cells scATAC-seq; Mus musculus; ATAC-seq
Nuclei were isolated and scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics;10xGenomics.com CG000496 Rev A). Briefly, after counting, nuclei concentrations were adjusted to the desired capture number 10,000 based on the number of available nuclei and the desired multiplet rate (described on 10X protocol). To minimize potential multiplets, we typically aimed to capture <6,000 nuclei per channel. Next, 5 μl of the resulting resuspended nuclei were combined with ATAC Buffer and ATAC Enzyme (10x Genomics PN-1000390) to form a transposition mix, which was then incubated for 60 min at 37 °C.  A master mix composed of Barcoding Reagent, Reducing Agent B and Barcoding Enzyme (10X PN-1000390) was then added to the same tube as transposed nuclei. The resulting solution was loaded onto a Chromium Chip H(10x Genomics; PN-1000161). Vortexed Chromium Single Cell ATAC Gel Beads and Partitioning Oil were also loaded onto the same Chromium Chip H before attaching a 10x Gasket and placed. into a Chromium Single Cell Controller instrument (10X genomics). Resulting single-cell GEMs were collected at the completion of the run (~17 min) and linear amplification was performed on AB cycler: 72 °C for 5 min, 98 °C for 30 s, cycled 14×: 98 °C for 10 s, 59 °C for 30 s and 72 °C for 1 min. Emulsions were coalesced using the Recovery Agent (10x Genomics; 220016), then subjected to Dynabeads (2000048) and SPRIselect reagent (Beckman Coulter;B23318) bead clean-ups. Indexed sequencing libraries were constructed by combining the barcoded linear amplification product with a sample index PCR mix comprising SI-PCR Primer B, Amp Mix and Chromium i7 Sample Index Plate N, Set A (10x Genomics; 3000262). Amplification was performed on an AB cycler: 98 °C for 45 s, for 12 cycles: 98 °C for 20 s, 67 °C for 30 s, 72 °C for 20 s, with a final extension of 72 °C for 1 min. The sequencing libraries were subjected to a final bead clean-up SPRIselect reagent and quantified by Qubit and TapeStation HS1000. Sequencing libraries were loaded on an Illumina Novaseq sequencer with 2 × 50 paired-end kits using the following read length: 51 bp read 1N, 8 bp i7 index, 24 bp i5 index (trimmed later on) and 51 bp read 2N using a loading molarity of 280 pM (Standard loading) or 180 pM (Xp loading).