Bioanalyzer dna1000 chips

RNA was isolated from the sorted NK cells using the RNeasy^® Mini Kit (205113, Qiagen). RNA integrity was verified with the Agilent Bioanalyser. DNase-treated RNA was treated for library preparation using the Truseq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer’s instructions. An initial poly(A) RNA isolation step (included in the Illumina protocol) is performed on 10 ng of total RNA to keep only the polyadenylated RNA fraction and remove the ribosomal RNA. A step of fragmentation is then performed on the enriched fraction, by divalent ions at high temperature. The fragmented RNA samples were randomly primed for reverse transcription followed by second-strand synthesis to create double-stranded cDNA fragments. No end repair step was necessary. An adenine was added to the 3′-end and specific Illumina adapters were ligated. Ligation products were submitted to PCR amplification. The obtained oriented libraries were controlled by Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified by spectrofluorimetry (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen). Sequencing was performed on the Illumina Hiseq2500 platform to generate single-end 100 bp reads bearing strand specificity.

RNA was isolated from the sorted cell populations using the Rneasy^® Mini Kit (205113, Qiagen). RNA integrity was checked using the Agilent Bioanalyzer System. Dnase-treated RNA was treated for library preparation using the Truseq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer’s instructions. An initial poly(A) RNA isolation step (included in the Illumina protocol) is performed on 10 ng of total RNA to keep only the polyadenylated RNA fraction and remove the ribosomal RNA. A fragmentation step is then performed on the enriched fraction, by divalent ions at high temperature. The fragmented RNA samples were randomly primed for reverse transcription, followed by second-strand synthesis to create double-stranded cDNA fragments. No end repair step was necessary. An adenine was added to the 3’-end and specific Illumina adapters were ligated. Ligation products were submitted to PCR amplification. The obtained oriented libraries were controlled by Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified by spectrofluorimetry (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen). Sequencing was performed on the Illumina Hiseq2500 platform to generate single-end 100 bp reads bearing strand specificity.

All samples were processed simultaneously. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, San Jose, CA) was used for cDNA synthesis with an input of 150 pg total RNA. Amplified cDNA was visualized and quantified using BioAnalyzer High Sensitivity chips (Agilent Technologies, Santa Clara, CA). Three hundred picograms of purified cDNA were brought up to 15 uL in volume and sheared on the Covaris LE220 (Covaris Inc., Woburn, MA) using the shearing parameters of PIP 180, 20% DF, 50 bursts/cycle, for 270 seconds with Y-dithering of 5 mm at 20 mm/s. Sequencing ready libraries were generated with 10 uL of sheared cDNA (200 pg) using the ThruPLEX DNA-Seq Library Preparation Kit (Takara Bio, San Jose, CA) and assessed for quality on BioAnalyzer DNA1000 chips (Agilent Technologies, Santa Clara, CA). Libraries were quantified using the Kapa Quant Kit for Illumina sequencing (Kapa Biosystems, Wilmington, MA), normalized to 4 nM, pooled equally, and prepared for sequencing on the NextSeq (Illumina, San Diego, CA) following the user manual guidelines for High Output single read 75 cycle chemistry runs.

RNA was isolated as described above. Polyadenylated messenger RNA was purified from 1 μg RNA and the library was generated using the TruSeq stranded mRNA library prep Kit, set A and B (Illumina; #RS-122-2101 and #RS-122-2102). The obtained directional libraries were controlled by Bioanalyzer DNA1000 Chips (Agilent; # 5067-1504) and quantified by spectrofluorimetry (QuantiT High-Sensitivity DNA Assay Kit, #Q33120; Invitrogen).

Amplicon PCR was performed with microbial genomic DNA using a concentration of 5 ng/µl in 10 mM Tris pH 8.5. PCR amplification of the V3/V4 regions of bacterial 16S rRNA encoding gene was carried out using the primers Pro341-XT (TCG-TCG-GCA-GCG-TCA-GAT-GTG-TAT-AAG-AGA-CAG-CCT-ACG-GGN-BGC-ASC-AG) and Pro805-XT (GTC-TCG-TGG-GCT-CGG-AGA-TGT-GTA-TAA-GAG-ACA-GGA-CTA-CNV-GGG-TAT-CTA-ATC-C) which resulted in amplicon sizes smaller than 550 bp. The details of library constructions, such as index PCR, PCR clean-up 2, library quantification, normalization, and pooling were performed corresponding to the Illumina “16S Metagenomic Sequencing Library Preparation” protocol.
Bioanalyzer DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and Qubit kits (Thermo Fischer Scientific, Waltham, MA, USA) were applied for quantity and quality controls of each individual sample library and the final library pool; 5 pM of the final library mixture, containing at least 5% PhiX control, was exposed to one individual sequencing run using a 2x250 or 2x300 cycle 3 reagent cartridge on an Illumina MiSeq machine. All raw data fastq files were used for sequence data analyses.

We used 2 to 5 μg of large RNA to purify polyadenylated mRNAs and to build an RNA library, using TruSeq RNA Sample Prep Kit v2 (Illumina, #RS-122-2001 and #RS-122-2002, San Diego, CA) as recommended by the manufacturer. The nondirectional libraries thus obtained were CTRL led by Bioanalyzer DNA1000 Chips (Agilent Technologies, #5067–1504, Santa Clara, CA) and quantified using spectrofluorimetry (Quant-iT™ DNA High-Sensitivity Assay Kit, #Q33120, Invitrogen, Life Technologies, Carlsbad, CA).