High sensitivity dna kit

For small RNA sequencing (small RNA-seq), 5 μL of extracted RNA was used as starting material to generate cDNA barcoded libraries. Library preparation was performed using the Illumina TruSeq Small RNA Library Prep Kit v2 (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, specific adapters were ligated to the 5′ and 3′ ends of RNA molecules and used as templates for reverse transcription. The synthesized cDNA fragments were subsequently amplified by 15 cycles of polymerase chain reaction using unique index primers. The obtained libraries were quantified on Bioanalyzer 2100 with High Sensitivity DNA Kit (Agilent Technologies, Inc., Santa Clara, CA, USA), pooled together and run on 6% Novex TBE-PAGE gel (Thermo Fisher Scientific, Waltham, MA, USA) for size selection. cDNA fragments of 140–160 bp in size were excised from the gel and purified. A concluding Bioanalyzer 2100 run with a High Sensitivity DNA Kit (Agilent) was performed to verify the length range of the library and calculate the final working solution concentration. Small RNA-seq was carried out according to Illumina pipeline on NextSeq 500 Instrument (Illumina) with the Next Seq High Output kit v2 (75 cycles) (Illumina).

The “TruSeq^® small RNA library Prep” kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries for small RNA sequencing from a maximum of 1μg of total RNA isolated from the whole blood of patients with KTx according to manufacturer’s instruction. RNA quality was assessed on a bioanalyzer using the RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany) and only RNA with a RIN >8 was considered for further processing. After quality control (High sensitivity DNA Kit, Agilent Technologies, Waldbronn, Germany) the resulting cDNA libraries were purified by gel-electrophoresis for the small RNA containing cDNA fraction at ~150 bp. The final small RNA cDNA libraries were quality checked (High sensitivity DNA Kit, Agilent Technologies, Waldbronn, Germany) and quantified (Qubit dsDNA HS Assay Kit, Invitrogen, Darmstadt, Germany). The final libraries were single-end (50 bp) sequenced on a HiSeq2500 Illumina Next Generation Sequencing Device (Illumina, San Diego, CA, USA). MicroRNAs were identified and quantified using miRDeep2.0.0.8 [16 (link)]. Differential expression analyses were performed using DESeq2 [17 (link)]. MicroRNAs with p-values below 0.05 and an absolute fold change ≥1.5 were considered significantly differentially expressed. Principal component analyses were based on rlog-normalized reads.

We performed virus infection assays using MØ from 20 different donors and RNAseq studies were carried out with 4 representative samples. Purified MØ populations (HSAneg and HSApos) were resuspended and frozen in QIAzol lysis buffer. Total RNA was isolated from sorted cell populations using the miRNeasy Kit from QIAGEN (Valencia, CA). RNA-seq libraries were prepared using the Scriptseq V2 complete gold kit (Epicentre) and the TrueSeq RNA Library Prep Kit V2 (Illumina) following manufacturer’s instructions. RNA integrity was assessed using RNA 6000 nano and pico kits. DNA libraries were quantified using high sensitivity DNA kits following manufacturer’s instructions (Agilent). Details for the bioinformatics analysis are described in the supplemental experimental procedures.

LifeFeng Genomic DNA Purification Kits (Lifefeng Biotech Co., Ltd., Shanghai, China) were used to extract genomic DNA from peripheral blood samples. DNA concentration and quality were examined by NanoDrop2000 (Thermo Scientific, USA). Twenty-three pairs of primers divided into two pools were designed covering all exons, UTRs, and exon–intron boundary of BNIP3L gene. The sequences of primers and their targeted regions are shown in Table S1. Two-staged PCR process was performed for library construction. All the PCR reagents and protocol were supported by Shanghai DYnastyGene Company. High Sensitivity DNA kits and 2100 Bioanalyzer (Agilent Technologies, USA) were used to determine the size distribution of DNA library fragments. The Illumina HiSeq X Ten System (Illumina, USA) was used to sequence the final DNA libraries as PE 150 bp reads.

Fluidigm C1 Single-Cell Integrated Fluidic Circuit (IFC) and SMARTer Ultra Low RNA Kit were used for single-cell capture and complementary DNA (cDNA) generation. cDNA quantification was performed using Agilent High Sensitivity DNA Kits and diluted to 0.15–0.30 ng/μL. The Nextera XT DNA Library Prep Kit (Illumina) was used for dual indexing and amplification with the Fluidigm C1 protocol. Ninety-six scRNA-seq libraries were generated from each tumor/Cd11b + sample and subsequently pooled for 96-plex sequencing. cDNA was purification and size selection was carried out twice using 0.9X volume of Agencourt AMPure XP beads (Beckman Coulter). The resulting cDNA libraries were quantified using High Sensitivity DNA Kits (Agilent).