Nuclei isolation for Single-Cell ATAC-Seq was performed according to the manufacturer’s manual (Chromium Nuclei Isolation Kit, 10x Genomics). Briefly, frozen hippocampal tissue was homogenized in 500 μl of pre-chilled lysis buffer using Dounce homogenizers. The homogenate was then transferred into pre-chilled Nuclei Isolation Columns, and the columns were centrifuged at 16000*g for 30 seconds at 4 °C. The flowthrough was briefly vortexed for 10 seconds, followed by centrifugation at 500*g for 3 min at 4 °C. The nuclear pellet was resuspended in 500 μl of Debris Removal Buffer and centrifuged at 700*g for 10 min at 4 °C. The nuclear pellet was then resuspended in 1 ml of Wash Buffer, centrifuged at 500*g for 5 min at 4 °C, and finally resuspended in 50 μl of Resuspension Buffer. The Single Cell ATAC-Seq protocol, comprising of the Transposition, GEM Generation, Barcoding, and Library Construction steps were carried out according to the manufacturer’s protocol (Chromium Next GEM Single Cell ATAC Kit, 10x Genomics). Libraries were sequenced in paired end in Illumina Novaseq.
Chromium next gem single cell atac kit
Manufactured by 10x Genomics
The Chromium Next GEM Single Cell ATAC Kit is a product offered by 10x Genomics for single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis. The kit enables the capture and barcoding of single cells for the purpose of studying chromatin accessibility.
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Lab products found in correlation
2 protocols using chromium next gem single cell atac kit
1
Nuclei Isolation for Single-Cell ATAC-Seq
2
Profiling Chromatin Accessibility in Mouse Islets
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Mouse islets were freshly isolated at CT4 and CT16 (n = 2 mice per CT and group), >100 islets were counted, and nuclei isolation was performed as noted above using ice-cold 1× lysis buffer from 10X Genomics (Pleasanton, CA). A portion of isolated nuclei were stained with Trypan Blue for manual counting and to ensure a single-nucleus suspension before library preparation. Approximately 5000 nuclei per sample were subsequently aliquoted for scATAC-seq library preparation using the Chromium Next GEM Single-Cell ATAC Kit (10X Genomics). Ten microliters of 1× Tn5 Transposition enzyme was mixed with 5 μl of the diluted nuclei suspension and was incubated at 50°C for 1 hour. The tagmented nuclei were partitioned into Gel Bead-In Emulsions (GEMs) and barcoded by unique molecular identifiers using the Chromium Next GEM Single-Cell ATAC library and Gel Bead kit and the C1 Chromium Instrument (10X Genomics). scATAC-seq libraries were then prepared and indexed using the Chromium i7 Sample Index kit (10X Genomics) with library size determined by Fragment Analysis (Agilent) before sequencing. Paired-end, dual-indexed sequencing of the libraries (50-bp read #1, 8-bp i7 index, 16-bp i5 index, and 50-bp read#2) was performed using an Illumina HiSeq instrument. scATAC-seq bioinformatic analysis was performed as described in the Supplementary Materials.
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