Novaseq 6000

Chromium Controller platform of 10X Genomics was used for single-cell partitioning and barcoding. Single Cell 3’ Gene Expression were prepared according to the manufacturer’s protocol “Chromium NextGEM Single Cell 3’ Reagent Kits v3.1” (CG000315, 10X Genomics). A NovaSeq 6000 Illumina sequencing system was used for paired end sequencing of the Single Cell 3’ Gene Expression libraries at a sequencing depth of ~17.000 mean reads per cell for control liver and 35.000 mean reads per cell for Nras^G12D/Pten^KO. NovaSeq 6000 paired end sequencing was performed using NovaSeq SP Reagent Kit v1.5 (cat# 20028401, Illumina) and NovaSeq S1 Reagent Kit v1.5 (cat# 20028319, Illumina).

RNA-sequencing was performed on 3 keloid specimens as well as the patients’ normal skin (at least 1 cm from the keloid lesion). These 3 patients were enrolled between March 2021 and April 2021. All of them signed informed consent forms. And this study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (No. 2021-SR-418). Keloid and the surrounding 1cm of normal tissue were resected by the same surgeon. TruSeq chain mRNA Library Prep Kit (Illumina) was used to generate the Library. Next-generation sequencing was performed on Illumina NovaSeq6000 (Illumina Inc., 100 cycles, single-read sequencing). Sample quality was assessed by FastQC. Illumina NovaSeq6000 analyzed RNA sequencing data for more global analysis of genomic abnormalities. Data is preprocessed using standard pipelines that contain quality control indicators, such as FastQC and MultiQC. Sequence alignment based on STAR-RNA sequencer and sequencing reads allocated to genome features via Featurecots and Vomo-Transformed.

We established two libraries: the chain-specific library and the small RNA library. For lncRNA, mRNA and circRNA sequencing, the chain-specific library was constructed using the TruSeq Stranded Total RNA Library Prep Plant/Gold/Globin +NEBNext^® Ultra Directional RNA Library Prep Kit (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions. Then the chain-specific library was subjected to PE150 (paired-end 150nt) sequencing on the Illumina NovaSeq 6000 (Novogene, Sacaramento, CA, USA). For miRNA sequencing, the small RNA library was constructed using the NEB Next^® Multiplex Small RNA Library Prep Set for Illumina^® (Set 1) (NEB, Ipswich, MA, USA) following manufacturer’s instructions. Subsequently, the small RNA library was sequenced on Illumina NovaSeq 6000 (Novogene, Beijing, China), and 50 bp single-end reads were generated. Raw sequence files have been deposited at NCBI’s Gene Expression Omnibus (Accession code: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190744).

In this study, the E. faecium B13 strain genome was sequenced by a combination of PacBio RS II and Illumina NovaSeq 6000, respectively.
For Illumina pair-end sequencing of this strain, purified genomic DNA was sheared into smaller fragments with 300~500 bp by Covaris M220 (Covaris, Woburn, MA, USA), and genomic libraries of E. faecium B13 were constructed using the TruSeq™ Nano DNA Sample Prep Kit (Illumina, San Diego, CA, USA). The whole genome sequencing was performed on an Illumina NovaSeq 6000 (150 bp*2, Shanghai BIOZERON Co., Ltd., Shanghai, China) using the Truseq SBS Kit (Illumina, California, USA) with 300 cycles.
Moreover, genomic DNA was processed into 15–20 kb fragments by the G-tubes method and sequenced using a PacBio BS(Sequel) II instrument(PacBio, Menlo Park, CA, USA) following the Pacbio standard protocol. The data were assembled using unicycler version 0.4.8. The protein sequences were predicted by GeneMarkS (version 4.17), and the COG database was used to annotate the functions of the predicted open reading frames.

An Illumina paired-end library was constructed using the TruSeq Nano DNA HT Sample Preparation Kit (Catalog No. TG-202-1003, Illumina, San Diego, CA) according to the manufacturer’s instructions, and sequenced by Illumina NovaSeq 6000 (Illumina, San Diego, CA). Three paired-end CCS libraries with the small insert size of 15 kb were constructed, and sequenced using the PacBio Sequel II platform (PacBio, San Francisco, CA). High-fidelity (HiFi) reads were generated by CCS software (https://github.com/PacificBiosciences/ccs). To improve the allele-aware genome to chromosome scale, two Hi-C libraries were constructed and sequenced by Illumina NovaSeq 6000 using DNA extracted from the same individual.

The genomic DNA of T. mongolicus was extracted from the leaves using a modified CTAB method [34 (link)]. Quality of the isolated DNA was assessed using NanoDrop-2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and Qubit 3.0 fluorometer (Life Technologies). The genomic DNA was sheared to an average size of 500 bp, and a library with an insert size of 500 bp was prepared using the PairedEnd DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). The library was sequenced using the Illumina NovaSeq6000 platform (Illumina Inc., San Diego, CA, USA) for genome survey and assessment. For genome sequencing, DNA fragments > 20 kb were selected for library preparation using BluePippin (SAGE). The PacBio library was prepared using the SMRTbell Template Prep Kit-SPv3 following the manufacturer’s instructions (Pacific Biosciences, Menlo Park, CA, USA) and was sequenced on the Pacific Biosciences Sequel system (Pacific Bioscience, CA, USA). The Hi-C library [35 ] was constructed using the HindIII restriction enzyme as per the manufacturer’s instructions (BioMarker Technologies Company) [36 (link)] and was sequenced on Illumina NovaSeq6000 platform for chromosome construction. For RNA-seq used in genome assembly and annotation, libraries were constructed for the root, stem, leaf, and flower of T. mongolicus using a paired-end model and were sequenced on Illumina NovaSeq6000 platform.