Revision as of 10:16, 13 August 2019

Prep Descriptions

KAPA HyperPrep mRNA

Libraries were prepared for RNA-Seq using KAPA Biosystems KAPA mRNA HyperPrep Kit, according to manufacturer’s directions. Briefly, total RNA (0.1-1 ug) is enriched for polyadenylated sequences using oligo-dT magnetic bead capture. The enriched mRNA fraction is then fragmented, and first-strand cDNA is generated using random primers. Strand specificity is achieved during second-strand cDNA synthesis by replacing dTTP with dUTP, which quenches the second strand during amplification. The resulting cDNA is A-Tailed and ligated with indexed adapters. Finally, the library is amplified using a DNA Polymerase which cannot incorporate past dUTPs, effectively quenching the second strand during PCR.

TruSeq PolyA

Libraries were prepared for RNA-Seq using Illumina’s TruSeq Stranded mRNA Library Preparation Kit according to manufacturer’s directions. Briefly, total RNA (0.1-1 ug) is enriched for polyadenylated sequences using oligo dT magnetic bead capture. The enriched mRNA fraction is then fragmented, and first-strand cDNA is generated using random primers. Strand specificity is achieved during second-strand cDNA synthesis by replacing dTTP with dUTP, which quenches the second strand during amplification. The resulting cDNA is A-Tailed and ligated with indexed adapters. Finally, the library is amplified using a DNA Polymerase which cannot incorporate past dUTPs, effectively quenching the second strand during PCR.

TruSeq RiboZero

Libraries were prepared for RNA-Seq using Illumina’s TruSeq Stranded Total RNA Library Preparation Kit according to manufacturer’s directions. Briefly, total RNA (0.1-1 ug) is ribo-depleted using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. The enriched fraction is then fragmented, and first-strand cDNA is generated using random primers. Strand specificity is achieved during second-strand cDNA synthesis by replacing dTTP with dUTP, which quenches the second strand during amplification. The resulting cDNA is A-Tailed and ligated with indexed adapters. Finally, the library is amplified using a DNA Polymerase which cannot incorporate past dUTPs, effectively quenching the second strand during PCR.

SMARTer V4

Upto 10 ng of sample was prepared using Takara’s SMART-Seq v4 Ultra Low Input RNA protocol per manufacturer’s guidelines. Briefly, RNA underwent reverse transcription via template switching and amplification, resulting in double stranded cDNA. The amount of cDNA between 100-3000 bp was calculated by Fragment Analyzer and Qubit. 150pg of the sample in range was then added to Illumina’s Nextera XT and processed according to manufacturer’s guidelines. Briefly, the cDNA underwent tagmentation, using Nextera XT’s transposase, and unique dual indexes were added by PCR amplification. Final libraries went through QC with the Fragment Analyzer and qPCR on a Roche Light Cycler 480 II. All libraries were then pooled and sequenced at single-end 40 base-pair using the Illumina HiSeq 2500. Demultiplexed sequencing data was handed over.

Sequencing

All libraries are qPCR'ed using KAPA qPCR library quant kit as per manufacturers protocol. The samples are loaded on the HiSeq 2500 based on qPCR concentrations.

Data Processing

All data is preprocessed and converted to FASTQ format. Please refer to the section Sequencing Format for details. Format of FASTQ can be converted using the script available under Scripts.

Single cell 10x data is processed using cellranger pipeline and hence the above links do not apply to it.