Agilent 2100 bioanalyzer dna1000 chip

The RNA was quantified using Qubit instrument (Invitrogen, Carlsbad, CA, USA) and RNA BR assay kit (Invitrogen). 100 ng of RNA was used as input for library preparation using TruSeq RNA Access Library preparation kit (Illumina, San Diego, CA, USA) as per the manufacturer’s instructions. Briefly, the RNA was fragmented into small pieces under high temperature using divalent cations. The RNA fragments were immediately reverse transcribed to first strand cDNA using random hexamers. Following the first strand, second strand was synthesized by incorporating dUTP instead of dTTP. The sequencing adaptors were ligated to the double-stranded cDNA followed by a single “A” nucleotide adenylation at 3’ end of blunt fragments. The final library was created by capturing the coding regions of the transcriptome using sequence-specific probes. The yield of cDNA libraries was quantified using Qubit dsDNA HS assay kit (Invitrogen) and size distribution of the cDNA libraries were determined using the Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies). The clusters were generated on a cBot cluster generation system (Illumina) and sequencing was done on Hiseq 4000 with 300 bp paired-ends.

Only the small 3rd metatarsal 3150 from layer 4c of Grotta Paglicci (direct ¹⁴C date: 14,372–13,759 cal. yr bp) was selected for aDNA analysis. The oldest specimens are too small to extract enough quantity of bone powder without significantly damaging them. DNA analysis was carried out in the Molecular Anthropology Laboratory of the University of Florence, exclusively dedicated to ancient DNA analysis. Blanks as negative controls were used in all of the experimental steps to monitor the absence of contaminants in reagents and environment. To remove potential contamination, the outer layer of the bone was mechanically taken out using a dentist drill with disposable tip. After brushing, sample was irradiated by ultraviolet light for 45 min in a Biolink DNA Crosslinker (Biometra). The DNA was extracted from approximately 50 mg of bone powder following a published silica-based protocol^{50 (link),80 (link)} and eluted in 100 µl of TET buffer (10 nM Tris, 1 mM EDTA and 0.05% Tween-20). 20 μl of DNA extract were transformed into genetic library following a double-stranded DNA protocol^{81 (link)} using a unique combination of two indexes. Sample and negative controls were checked with Agilent 2100 Bioanalyzer DNA 1000 chip. Libraries were then enriched for mitochondrial DNA following a capture protocol^{81 (link),82 (link)} and sequenced on an Illumina MiSeq run for 2 × 75 + 8 + 8 cycles.

We used a starting input material of 100 ng of RNA for the library preparation using TruSeq RNA Access Library preparation kit (RS-301-2001 and RS-301-2002, Illumina, San Diego, CA, USA) as per the manufacturer's instructions. Briefly, the RNA was fragmented into small pieces under high temperature using divalent cations. The RNA fragments were immediately reverse transcribed to first-strand cDNA using random hexamers. Following the first strand, the second strand was synthesized by incorporating dUTP instead of dTTP. The sequencing adaptors were ligated to the double-stranded cDNA followed by a single "A" nucleotide adenylation at the 3' end of blunt fragments. The final library was created by capturing the regions of the transcriptome using sequence-specific probes. The yield of cDNA libraries was quantified using the Qubit dsDNA HS assay kit (Q32855, Invitrogen), and the size distribution of the cDNA libraries was determined using the Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies). The clusters were generated on a cBot cluster generation system (Illumina), and sequencing was done on Hiseq 4000 with 150 bp paired-ends.

Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). Digested DNA segments were further sheared by Bioruptor (Diagenode) to ∼300 bp. After purification by AMPure XP beads (Beckman Coulter, Cat. no. A63880), DNA segments were end-repaired, followed by 3′-adenylation and adaptor ligation according to standard Illumina TruSeq DNA sample preparation procedures. Adaptor-ligated DNA segments were PCR amplified for 15 cycles, purified by AMPure XP beads and suspended in 20 μl of resuspension buffer to yield the sequencing library. Quality control and concentration measurement were performed using Agilent 2100 Bioanalyzer DNA 1000 Chip and qPCR. Library was sequenced by using Illumina HiSeq 2500.

The quality of RNA was evaluated by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with the RNA 6000 Nano LabChip Kit. Samples with RNA integrity number (RIN) greater than 7 were used for library preparation. RNA was quantified by Qubit fluorometer and the Qubit RNA broad range (BR) assay Kit (Thermo Fisher Scientific). For library preparation, 100 ng of RNA and Illumina^® TruSeq Stranded RNA Library Preparation Kit (Illumina, Foster City, CA, USA) were used. Briefly, RNA was purified using poly-oligo beads, and fragmented into small pieces under high temperature by divalent cation. Using random hexamers, fragments of RNA were reverse transcribed to first-stranded cDNA. The second strand of cDNA was synthesized by incorporating dUTP instead of dTTP. The adaptors were then ligated to the double-stranded cDNA, followed by a single “A” nucleotide adenylation at 3′ end of blunt fragments. The Qubit dsDNA high sensitivity (HS) assay kit (Thermo Fisher Scientific) was used to determine the yield of DNA libraries, and Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies) was used to determine the size distribution of the DNA libraries. The clusters were generated by cBot, and sequencing was done on a HiSeq 4000 system with 300 bp paired-end, using the HiSeq 3000/4000 SBS Kit (Illumina).