High-molecular weight DNA from the Hi5 cells (extracted using the Bionano Plug Lysis protocol) was also used to make 10X libraries, as per the Chromium Genome library preparation protocol from 10X Genomics (Pleasanton, CA, USA). In brief, 0.9 ng/µL DNA were used for GEM generation in the Chromium Controller machine (10X Genomics, Pleasanton, CA, USA). The long DNA molecules were partitioned along with oligo-coated Gel Beads that provide a 16 bp 10X barcode, an Illumina R1 sequence, and a 6 bp random primer sequence. Isothermal incubation of the GEMs at 30 °C for 3 h, followed by 65 °C for 10 min produced barcoded fragments. These fragments were recovered from the GEMs and cleaned up for subsequent library preparation steps that included end repair, A-tailing and adapter ligation per the manufacturer’s recommendations. Eight cycles of amplification during the sample index PCR provided enough yield of the indexed library. The library was quantitated by qPCR and sequenced on NextSeq (High output kit) (Illumina, San Diego, CA, USA) with 2 × 150 paired-end reads.
Chromium controller machine
Manufactured by 10x Genomics
Sourced in United States
The Chromium Controller machine is a core component of the 10x Genomics platform. It is a versatile piece of laboratory equipment designed to automate the partitioning of biological samples into nanoliter-scale gel beads. This process allows for the high-throughput analysis of individual cells or molecules, enabling researchers to gain unprecedented insights into complex biological systems.
Automatically generated - may contain errors
2 protocols using chromium controller machine
1
10X Genomic Library Preparation from Hi5 Cells
2
Single-cell analysis of metastatic MDA-MB-231 subclones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the heterogeneity of the parental MDA-MB-231 population, a cell suspension of MDA-MB-231 was prepared using the TrypLE Express Enzyme (#12604021, Thermo Fisher Scientific). Using a Chromium controller machine (#120223, 10x Genomics) and a Chromium single-cell gel bead kit (#1000075, 10x Genomics), 10,000 cells were captured in gel bead in emulsion. The complementary DNA was then isolated, and the library was prepared as recommended by the manufacturer for the 3′ V3 kit (#1000078, 10x Genomics). Sequencing runs were completed on a NextSeq machine (Illumina) with the following specifications: Read1, 28 cycles; Read2, 91 cycles; Index Read, 8 cycles.
To verify the purity of the metastatic barcoded subclones and to investigate whether they were genomically distinct, subclones 13, 2, 9, 29, and 3 were FACS-purified from lung metastases. The number of cells sorted for single-cell analysis varied between the subclones on the basis of their frequency. For the dominant subclones 13, 2, and 9, 20,000 cells were sorted. For the minor subclones 29 and 3, 7000 and 1000 cells were sorted, respectively.
To verify the purity of the metastatic barcoded subclones and to investigate whether they were genomically distinct, subclones 13, 2, 9, 29, and 3 were FACS-purified from lung metastases. The number of cells sorted for single-cell analysis varied between the subclones on the basis of their frequency. For the dominant subclones 13, 2, and 9, 20,000 cells were sorted. For the minor subclones 29 and 3, 7000 and 1000 cells were sorted, respectively.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!