Nebnext ultra rna library prep kit for paired end multiplexed sequencing library

Genomic DNA isolation and bulk DNA sequencing were performed as we described in our previous work [45 (link)]. Briefly, fresh tumors were surgically resected from these two patients. Each tissue was cut into two pieces, with one for further single-cell collection and the other for bulk sequencing. This procedure could maximally ensure that the single-cell and bulk sequencing data were generated from a close region of the tissue. Genomic DNA were extracted using the QIAamp DNA Mini Kit (QIAGEN). Exon libraries were constructed using the SureSelectXT Human All Exon V5 capture library (Agilent). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
For bulk RNA analysis, small fragments of tumor tissues were first stored in RNAlater RNA stabilization reagent (QIAGEN) after surgical resection and kept on ice to avoid RNA degradation. RNA of tumor samples were extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s specification. Libraries were constructed using NEBNext Poly (A) mRNA Magnetic Isolation Module kit (NEB) and NEBNext Ultra RNA Library Prep Kit for Illumina Paired-end Multiplexed Sequencing Library (NEB). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.

Total RNAs were extracted from the non-co-cultured and co-cultured THP-1 cells using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The qualities of total RNAs were measured by spectrophotometer (NanoDrop, Thermo Fisher, USA). Libraries were constructed using the NEBNext Poly(A) mRNA Magnetic Isolation Module kit (NEB, USA) and NEBNext Ultra RNA Library Prep Kit for Illumina Paired-end Multiplexed Sequencing Library (NEB, USA). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150 bp paired-end reads. The Bulk RNA-seq data were processed using the STAR-rsem pipeline. Read counts per gene were normalized to gene length and to the total read counts, and directly compared to the log-transformed UMI count per gene for the single-cell samples using the Pearson correlation analysis.