In the case of the human archaeological dental samples, whole genome sequencing of calculus utilized random primers to amplify and sequence a largely unbiased subset of the total DNA.
A new amplification procedure, developed recently by NuGEN, utilizes random primers in addition to oligo (dT) primers, thus alleviating the 3' end bias.
Double-stranded cDNA was synthesized from total RNA using Roche 1st strand cDNA synthesis kit (Cat # 04379012001; Roche) utilizing random hexamer primers.
RNA was used for first-strand cDNA synthesis using random primers and SuperScript II Reverse Transcriptase (Invitrogen).
cDNA was made using SuperScript II Reverse Transcriptase (Life Technologies; 18064) of random primers and treated with Ribonuclease H (Life Technologies 18021-0711).
First-strand cDNA was synthesized using random primers and MLV reverse transcriptase (TaKaRa Bio, Beijing, China).
cDNAs were synthesized using random primers and MultiScribeTM reverse transcriptase (Applied Biosystems, Foster, CA).
VMC was extracted, reverse transcribed with random hexamers, amplified using barcoded semi-random primers, and sequenced on the Illumina HiSeq2500 platform (2 × 150 bp) at varying sequencing depths yielding 36,974,536 total reads (Methods).
Subsequently, the cDNA was utilized for semi-quantitative PCR utilizing intron-spanning primers and optimized reaction conditions.
After total RNA extraction by RNeasy Plus Mini as recommended by the manufacturer (Qiagen, #28106), 1 μg/sample of total RNA was subjected to reverse transcription with SuperScriptVILO-Reverse Transcriptase (Invitrogen) for 10 min at 25 °C, 1 h at 42 °C and 5 min at 85 °C, utilizing 50 pmol hexameric random primers.
