Nebnext ultra dna library preparation kit

150 ng of each purified RT-PCR sample was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. The fragment ends of 50 ng of these DNA fragments were repaired using the NEBNext Ultra DNA Library Preparation kit (New England Biolabs), followed by addition of the adaptors by using the NEBNext Multiplex Oligos for Illumina kit (Dual Index Primers Set 1, New England Biolabs). The resulting fragments were size-selected at 300 to 400 bp using Agencourt AMPure XP bead sizing (Beckman Coulter). Afterwards, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. After the 2×250 bp MiSeq paired-end sequencing run, the data were base called and converted to Illumina FASTQ files (Phred +64 encoding).

Soil DNA was used for 16S rRNA gene amplification using V3 and V4 primer sets (V3F-5’ CCTACGGGNBGCASCAG3’; V4R-5’ GACTACNVGGGTATCTAATCC3’) (Takahashi et al., 2014 (link)). The amplified products were checked by running on 2% (w/v) agarose gel. The PCR products were purified for paired-end library preparation. Approximately 5 ng/μL of the PCR amplicon was used for library preparation using the NEB Next Ultra DNA library preparation kit (New England Biolabs, United Kingdom), as per the manufacturer’s protocol. Library size and quality were determined using Agilent 2200, TapeStation (Agilent Technologies, United States). High-throughput sequencing was performed using Illumina HiSeq 2500 platform with 2*250 cycle chemistry to obtain the metagenomic data.

Fifty milligram cerebral cortex tissue was as ChIP samples. ChIP was done with Simple ChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads; CST-9005S) following the manufacturer’s protocols. The immunoprecipitations used antiH3K9cr (PTM-516RM, 1:50) and normal Rabbit IgG is used as negative controls. Before CHIP-sequencing, sample fragments have to be 200–500 bp qualified by Agilent2200. Generate Illumina sequencing libraries from the NEBNext^®Ultra™DNA Library Preparation Kit (New England Biolabs) manufacturer’s Manual. The library quality was examined using an Agilent 2100 bioanalyzer followed by high-throughput 150-base pair end sequencing on the Illumina Novaseq 6000 sequencer. Then these clean reads were aligned to rat reference genome (UCSC rn5) using bowtie2 software (v2.2.4) with default parameters. Peak calling was performed with MACS software (v2.2.7.1). Differentially enriched regions were identified by diffReps software (v1.55.4). Then, the enriched peaks were annotated with the UCSC RefSeq database, and the peak information was connected with gene annotations.

Total RNA was extracted from sorted LSCs (lin⁻c-kit⁺Sca1⁻CD16⁺CD34⁺) from two full blown leukemia mice developed from MLL-AF9-transduced Hsf1^fl/flVav-Cre HSPCs and two full blown leukemia mice developed from MLL-AF9-transduced Hsf1^fl/fl HSPCs (duplicate per genotype), respectively. cDNA synthesis and amplification were performed using the SMARTer Ultra Low Input RNA Kit (Clontech) starting with 20 ng of total RNA per sample, following the manufacturer’s instructions. cDNA was fragmented with Q800R sonicator (Qsonica) and used as input for NEBNext Ultra DNA Library Preparation Kit (NEB). Libraries were sequenced on Illumina’s HiSeq2000 in single-read mode with a read length of 50 nucleotides producing 60–70 million reads per sample. Sequence data in fastq format were generated using the CASAVA 1.8.2 processing pipeline from Illumina. Differential expression analysis was performed using the DESeq2 v1.30.1 R package^{97 (link)}. We considered genes differentially expressed between 2 groups of samples when the DESeq2 analysis resulted in an adjusted p-value of <0.01 and the log2-fold change in gene expression greater than or equal to 1.5 or less than or equal to −1.5. Gene set enrichment analysis (GSEA) 3.0 was done according to the previous report using the software downloaded from GSEA website (http://software.broadinstitute.org)^{98 (link)}.

The detailed methodology for detecting and sequencing P. falciparum genome using selective whole genome amplification (sWGA) has been published by Aninagyei et al [24 (link)]. The sWGA reaction was performed using DNA template (≥ 5 ng). The reaction mix was made up of the following: template DNA, 1× bovine serum albumin, 1 mM dNTPs, 2.5 μM of each amplification primer, 1× Phi29 reaction buffer and 30 units of Phi29 polymerase enzyme (New England Biolabs). Isothermal amplification conditions were used (35 °C for 5 min, 34 °C for 10 min, 33 °C for 15 min, 32 °C for 20 min, 31 °C for 30 min, 30 °C for 16 h prior to denaturing Phi29 polymerase enzyme 65 °C. Ampure XP cleaned amplicons washed with 200 μL of 80% ethanol and eluted with elution buffer. Using the NEBNext® Ultra™ DNA library preparation kit (New England Biolabs), DNA libraries were prepared prior to sequencing on Illumina HiSeq 2500 DNA Sequencer. After sequencing, demultiplexing and fastq data files were generated automatically. Low quality reads were trimmed (Bioedit v7.2) and each dataset analysed independently by mapping sequence reads to the P. falciparum 3D7 reference genome using Burrows-Wheeler Aligner. Allelic analysis was done for Pfcrt, Pfdhfr, Pfdhps, Pfmdr1 and Kelch13 genes.

The hypervariable V3–V4 region of the prokaryotic 16SrRNA gene from the total bacterial DNA (10 ng) was amplified using the primers, viz. Pro341F (5′-CCTACGGGNBGCASCAG-3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC-3′) (Takahashi et al., 2014 (link)). The amplified product was gel-purified to remove non-specific amplification, if there is any. Metagenomic library preparation was done using the NEBNext Ultra DNA library preparation kit (New England Biolabs) using equimolar quantities of PCR amplicon (5 ng). The library quantity and quality were estimated in Agilent 2200 TapeStation. The sequencing was then performed on an Illumina HiSeq 2500 platform (2 × 300 paired-end sequencings) (AgriGenome Labs Private Limited, Kochi, India). The high-quality samples were refined and used for metagenomics analysis.

Water was used in negative controls and carried through all steps of the library preparation until the ddPCR and partly also to sequencing. Molecule numbers in negative controls were orders of magnitude lower than samples, with the exception of one NEB double-strand preparation (NTC1 in Table 1). It has been reported that standard library preparation methods (e.g., Illumina's TruSeq DNA sample preparation kit, no. 15026486 Rev. A, or NEB's NEBNext Ultra DNA library preparation kit, v1.1) are less suitable for the generation of libraries from highly degraded and low-quantity DNA because a background of around 1 × 10E⁸ molecules may be created mostly derived from adapter dimers and synthesis artifacts present in the adapter oligonucleotide [11 (link)]. The number of sequencing reads and mapped reads was assessed for three negative controls of single-strand library preparation.