Revision as of 11:02, 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.

10X - Single cell - 3p

Cells were processed using the 10X Genomics Chromium Controller with the Single Cell 3ʹ v3 Reagent Kit, according to manufacturer’s directions. Briefly, a target number of cells per library was roughly achieved by loading 1.6 times the target number of cells in suspension, along with barcoded beads and partitioning oil, into the Chromium Controller, in order to create GEMs (Gel Beads in Emulsion). The Chromium Controller combines individual cells, first strand master mix, and gel beads containing barcoded oligonucleotides into single-cell droplets for first strand cDNA synthesis, so that each cell is marked with its own unique barcode during reverse transcription. After first strand synthesis is complete, the emulsion is dissolved and the cDNA is pooled for bulk processing as a single sample. The sample is fragmented, end-repaired, A-tailed and ligated with universal adapters. A second sample barcode is then added during the PCR step, allowing for unique library identification. The end result is a single library representing one cell suspension, containing data for each individual cell. Samples were then sequenced using an Illumina HiSeq 2500 with a read length of 28x40 base pairs.

Genome core switched to v3 kit in May 2019. Some preps may have been done in v2 due to specific requests from users. Typically the lane annotation file's prep method column would indicate if v3 was used for the preps

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.