Single-cell capture and pre-amplification were conducted on a GemCode instrument (10× Genomics) according to the manufacturer’s ‘instructions (Chromium™ Single Cell 3’ Reagent Kit v2). The generated library was sequenced using the Illumina X10 platform and the generated sequencing reads were aligned and analyzed using the Cell Ranger Pipeline (10× Genomics). The raw count data of each single cell were deposited into the public database of the Genome Sequence Archive for Human (GSA-Human) under accession number HRA000928. Single-cell analysis was conducted using Seurat9 (link). The potential doublet of single-cell data was detected using DoubleFinder10 (link). A connectivity map was constructed according to a previous ligand-receptor dataset35 (link) using CellPhoneDB16 (link). GO analysis was conducted using http://geneontology.org . Gene set variation analysis (GSVA) was used to perform deconvolution analysis36 .
Gemcode instrument
Manufactured by 10x Genomics
Sourced in United States
The GemCode Instrument is a piece of lab equipment designed and manufactured by 10x Genomics. The core function of the GemCode Instrument is to generate single-cell barcoded libraries for various genomic applications, such as single-cell RNA sequencing and single-cell DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Irys platform, by Bionano Genomics (2 mentions)
Hiseq x platform, by Illumina (2 mentions)
Pacbio rs 2 platform, by Pacific Biosciences (2 mentions)
Single cell 3 reagent kit, by 10x Genomics (1 mentions)
Cell ranger pipeline, by 10x Genomics (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Gemcode gel bead and library kit, by 10x Genomics (1 mentions)
Nextseq instrument, by Illumina (1 mentions)
Cd45 microbeads, by Miltenyi Biotec (1 mentions)
Hiseq x ten system, by Illumina (1 mentions)
8 protocols using gemcode instrument
1
Single-cell Transcriptome Profiling and Analysis
2
GemCode-Based Single-Cell Sequencing
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The GemCode Instrument from 10x Genomics was used for DNA sample preparation, indexing and barcoding. Around 1 ng of input DNA with a 50-kb length was used for the GEM reactions during PCR, and 16-bp barcodes were introduced into droplets. Then, the droplets were fractured following purification of the intermediate DNA library. Next, we sheared DNA into 500-bp fragments for constructing libraries, which were finally sequenced on NovaSeq.
3
Single-Cell DNA Sequencing Protocol
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DNA sample preparation was conducted using a GemCode Instrument from 10× Genomics (Pleasanton, CA, USA). A DNA sample of 1 ng was used for the GEM reaction procedure based on PCR. The library was finally sequenced using the Illumina HiSeq X Ten platform.
4
FFPE Sample Sequencing and Alignment
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To compensate for the fragmented nature of samples preserved with FFPE, we prepared sequencing libraries for the primary tumor FFPE sample and matched normal FFPE sample using the GemCode Gel Bead and Library Kit (10X Genomics) and the GemCode instrument (10X Genomics). The barcoded libraries were sequenced on an Illumina NextSeq instrument, and the resulting BCL files were demultiplexed and converted to fastq files using bclprocessor (v1.2.0). The aligner function of Long Ranger (v1.2.0) was run to generate aligned bam files. For the FFPE samples, the barcoded nature of the linked reads was used solely to improve alignment of the sequence reads; no phasing was performed for these data as the quality of FFPE samples is not adequate to infer long range haplotypes. Read mapping and sequencing coverage statistics are listed in Additional file 1 : Table S2. The GATK (v3.3) DepthOfCoverage tool was used to calculate coverage metrics [24 (link)].
5
Hybrid Genome Assembly Protocols
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A 20 kb insert size PacBio library was built as previously described (Strijk et al., 2019 ). This library was sequenced on the PacBio RS II platform (Pacific Biosciences), yielding about 37 Gb of data (read quality ≥0.80, mean read length ≥7 Kb). 10× Genomics DNA sample preparation, indexing, and barcoding were done using the GemCode Instrument (10× Genomics). About 0.7 ng of very high molecular weight DNA (N50 ~165 kb, 3% of the DNA >500 kb) was used for GEM reaction procedure during PCR, and 16 bp barcodes were introduced into droplets. Then, the droplets were fractured following the purifying of the intermediate DNA library. Next, we sheared the DNA into 500 bp for fragments constructing libraries, which were then sequenced on the Illumina HiSeq X platform (Illumina Inc.), according to the manufacturer's instructions. The same high molecular weight DNA was used to construct a Bionano optical map using an Irys platform (BioNano Genomics) with the Nt. BspQ1 (8.19 labels/100 kb), of which 95.9 Gb (120.01×, Table 1 ) data were generated.
6
Lung Single-Cell Preparation Protocol
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Lung parenchymal tissue preparation and scRNA-Seq sample preparation were performed as previously described (23 (link), 24 (link)). In brief, human lungs specimens were first confirmed to be from the most distal portion of the lung through direct visualization of the pleural lining. The pleural lining was separated from the lung parenchyma and discarded followed by microdissection along the bronchovascular tree. Specimens were then mechanically minced prior to enzymatic digestion with collagenase, dispase and DNase. Samples were then filtered, RBC lysed with ACK lysis buffer,and processed into a single-cell suspension. CD45+ immune cells were depleted using MACS LS columns with CD45 microbeads (Miltenyi Biotec, 130-045-801) with 2 × 106 cells per column to enhance purity and viability. Following the single-cell suspension, the samples were loaded onto a GemCode instrument (10X Genomics) to generate single-cell barcoded droplets (GEMs) according to the manufacturer’s protocol.
7
Barcoded Sequencing for Scaffolding Assemblies
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We used a GemCode Instrument from 10× Genomics to prepare DNA samples and for automated barcoding. Approximately 1 ng of input DNA was used for the gel-bead-in-emulsion (GEM) reaction. Each input DNA fragment was encapsulated into a GEM and labeled with a unique 10× barcode. GEMs underwent isothermal incubation to generate 10×-barcoded amplicons. Differently barcoded amplicons were mixed and sheared into 500 bp to construct an NGS-ready library. The constructed library was finally sequenced on an Illumina HiSeq X Ten system. The linked reads of the 10× Genomics data were aligned to the contigs generated from PacBio data using bowtie2 with parameters (-D 1 -R 1 -N 0 -L 28 -i S,0,2.50 -n-ceil L,0,0.02 -rdg 5,10 -rfg 5,10 -no-unal), and fragScaff (v2-1)22 (link) was used to build scaffolds using the barcoded sequencing reads with parameters (-fs1 '-m 3,000 -q 30 -E 30,000 -o 60,000′ -fs2 '-C 5′ -fs3 '-j 1 -u 3′).
8
Multi-omics integration for high-quality genome assembly
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A 20 kb insert size PacBio library was built as previously described (Strijk et al. 2014) . This library was sequenced on the PacBio RS II platform (Pacific Biosciences, Menlo Park, CA, USA), yielding about 37 Gb of data (read quality [?] 0.80, mean read length [?] 7 Kb). 10X Genomics DNA sample preparation, indexing, and barcoding were done using the GemCode Instrument (10X Genomics, Pleasanton, CA, USA). About 0.7 ng of very high molecular weight DNA (>50 kb) was used for GEM reaction procedure during PCR, and 16 bp barcodes were introduced into droplets. Then, the droplets were fractured following the purifying of the intermediate DNA library. Next, we sheared the DNA into 500 bp for fragments constructing libraries, which were finally sequenced on the Illumina HiSeq X platform (Illumina Inc., San Diego, CA, USA), according to the manufacturer. A Bionano optical map was also constructed from Irys platform (BioNano Genomics, San Diego, CA, USA) from the same DNA, of which 95.9 Gb data were generated.
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