NCBISubmission

Prep Descriptions

Watchmaker mRNA Library Prep

Libraries were prepared for RNA-Seq using Watchmaker mRNA Library Prep Kit, according to manufacturer’s directions. The Watchmaker mRNA Library Prep Kit first enriches for mature, polyadenylated mRNAs using oligo dT beads. The enriched mRNA is then fragmented, and undergoes 1st and 2nd strand cDNA synthesis, incorporating dUTP to maintain strand information. The double stranded cDNA is then A-tailed, ligated to universal adapters, and amplified using indexed primers to allow sample multiplexing.

Watchmaker Total Library Prep with Qiagen FastSelect Ribodepletion

Ribodepletion is performed using QIAseq FastSelect kit (according to manufacturer's protocol). It works by preventing cDNA synthesis of unwanted rRNAs during library preparation. The FastSelect ribodepletion method uses species-specific* oligos, which are hybridized to the total RNA during a combined hybridization and fragmentation step. Following ribodepletion, watchmaker kit is used for the RNA that undergoes 1st and 2nd strand cDNA synthesis, incorporating dUTP to maintain strand information. The double stranded cDNA is then A-tailed, ligated to universal adapters, and amplified using indexed primers to allow sample multiplexing.

(*human, mouse, rat, plant, bacteria, fish, yeast, worm and fly)

Swift ChIP-Seq

Libraries were prepared for ChIP-Seq using Swift Science’s Accel-NGS Library Preparation Kit for Illumina Platforms according to manufacturer’s directions. The swift kit makes library from 10pg-100ng of double stranded input material. Briefly, the sample undergoes a series of incubations and purifications. The sample, through multiple incubations, repairs both 5’ and 3’ termini and sequentially attaches Illumina adapter sequences to the ends of fragmented dsDNA. The multiple bead-based clean-ups are used to remove oligonucleotides and small fragments, and to change enzymatic buffer composition between steps.

xGen Broad-Range RNA Library Prep Kit (IDT) - Formally Swift Std RNA Library Prep Kit

Libraries were prepared for RNA-Seq using IDT’s xGen Broad-Range RNA Library Prep Kit, according to manufacturer’s directions. First, total RNA (10 ng – 1 ug) is enriched for polyadenylated sequences using NEBNext poly(A) mRNA Magnetic Isolation Module (NEB). The enriched mRNA fraction is then fragmented, and first-strand cDNA is generated using random primers. Via an “Adaptase” step, simultaneous tailing and ligation add a “stubby adapter” to the 3’ ends of the cDNA. A subsequent extension step generates dsDNA, and then a second adapter is ligated onto the extension product. A final PCR amplification step adds indexes and sequences needed for flow cell binding. Strand specificity is achieved by bypassing the traditional second strand cDNA synthesis step prior to adapter ligation.

xGen Library Prep Kit (IDT) - Formally Swift Rapid RNA Library Prep Kit

Libraries were prepared for RNA-Seq using IDT’s xGen RNA Library Prep Kit, according to manufacturer’s directions. First, total RNA (100 ng – 1 ug) is enriched for polyadenylated sequences using NEBNext poly(A) mRNA Magnetic Isolation Module (NEB). The enriched mRNA fraction is then fragmented, and first-strand cDNA is generated using random primers with a “stubby adapter” overhang. An Exonuclease step removes excess RT primers and prevents them from becoming template downstream. Via an “Adaptase” step, simultaneous tailing and ligation add a second stubby adapter. A final PCR amplification step adds indexes and sequences needed for flow cell binding. Strand specificity is achieved by bypassing the traditional second strand cDNA synthesis step prior to adapter ligation.

KAPA HyperPrep mRNA

Libraries were prepared for RNA-Seq using Roche Diagnostics 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.

KAPA RNA HyperPrep Kit with RiboErase

Libraries were prepared for RNA-Seq using the KAPA Biosystems RNA HyperPrep Kit with RiboErase, according to manufacturer’s directions. Briefly, total RNA (0.1-1 ug) is ribo-depleted by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides. The enriched fraction is then fragmented with heat and magnesium, 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, and the cDNA is then A-Tailed. The final double strand cDNA is then 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 (Next GEM) - 3p v3 or v3.1

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. The 3’ beads contain a poly(dT) oligo that enables the production of barcoded, full-length cDNA from poly-adenylated mRNA. 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.

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

10X - Single cell (Next GEM) - 5p v2

Cells were processed using the 10X Genomics Chromium Controller with the Single Cell 5ʹ v2 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. The 5’ beads contain a template switching oligo that, combined with a poly(dT) primer, enables the production of barcoded, full-length cDNA from poly-adenylated mRNA. 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.

10X - Single Cell (Next GEM 5’) – V(D)J

Amplified full-length cDNA from mRNA generated using the Next GEM 5’ kit is used to amplify full-length V(D)J segments via PCR amplification with primers specific to either the TCR or BCR constant regions. Enzymatic fragmentation and size selection are used to generate variable length fragments that collectively span the V(D)J segments of the amplified TCR or BCR transcripts prior to library construction. The sample is then 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.

10X - Single Cell (Next GEM) - Multiome – 3’ GEX and ATAC

Nuclei were processed using the 10X Genomics Chromium Controller with the Next GEM Single Cell Multiome Reagent Kit, according to manufacturer’s directions. Briefly, nuclei suspensions are incubated in a Transposition Mix that includes a Transposase. The Transposase enters the nuclei and preferentially fragments the DNA in open regions of the chromatin. Simultaneously, adapter sequences are added to the ends of the DNA fragments. Transposed nuclei are combined with barcoded beads and partitioning oil into the Chromium Controller, in order to create GEMs (Gel Beads in Emulsion). Single Cell Multiome ATAC + GEX Gel Beads include both a poly(dT) sequence that enables production of barcoded, full-length cDNA from poly-adenylated mRNA for the gene expression library, and a Spacer sequence that enables barcode attachment to transposed DNA fragments for the ATAC library. A pre-amplification step provides template material for both downstream ATAC and gene expression library production.

To produce the ATAC library, P7 and a sample index are added to pre-amplified, transposed DNA during ATAC library construction via PCR.

To produce the 3’ gene expression library, barcoded, full-length pre-amplified cDNA is amplified again via PCR to generate sufficient mass for gene expression library construction. The sample is then 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.

Sequencing

All libraries are qPCR'ed using KAPA qPCR library quant kit as per manufacturers protocol. The samples are loaded on the HiSeq 2500/NovaSeq 6000 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.