NCBISubmission - Revision history

Sgupta at 20:46, 9 December 2025

2025-12-09T20:46:16Z

Sgupta at 19:44, 30 October 2024

2024-10-30T19:44:03Z

Sgupta at 19:41, 30 October 2024

2024-10-30T19:41:41Z

Sgupta at 14:26, 25 September 2023

2023-09-25T14:26:37Z

Sgupta at 13:56, 4 August 2023

2023-08-04T13:56:32Z

Sgupta: /* 10X Next GEM- Multiome – 3’ GEX and ATAC */

2023-08-04T13:55:55Z

‎10X Next GEM- Multiome – 3’ GEX and ATAC

Sgupta: /* 10X Next GEM 5’ – V(D)J */

2023-08-04T13:55:36Z

‎10X Next GEM 5’ – V(D)J

Sgupta at 13:54, 4 August 2023

2023-08-04T13:54:46Z

Sgupta at 13:39, 4 August 2023

2023-08-04T13:39:38Z

Sgupta at 18:54, 13 July 2023

2023-07-13T18:54:02Z

← Older revision		Revision as of 20:46, 9 December 2025
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	= Sequencing =		= 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.	+	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/AVITI based on qPCR concentrations. The sequencer used would be listed in the lane annotation excel delivered with the data along with the read length information.

@@ Line 3: / Line 3: @@
 === Watchmaker mRNA Library Prep ===
-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.
+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 ===
-QIAseq FastSelect 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. The RNA then 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.
+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)

← Older revision		Revision as of 19:41, 30 October 2024
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	= Prep Descriptions =		= Prep Descriptions =
		+
		+	=== Watchmaker mRNA Library Prep ===
		+
		+	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 ===
		+
		+	QIAseq FastSelect 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. The RNA then 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 ===		=== Swift ChIP-Seq ===

← Older revision		Revision as of 14:26, 25 September 2023
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	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.		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 ===		=== KAPA HyperPrep mRNA ===

@@ Line 26: / Line 26: @@
-=== 10X - Single cell - 3p v3 or v3.1 ===
+=== 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.
@@ Line 33: / Line 33: @@
 '''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 - 5p v2 ===
+=== 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.

@@ Line 40: / Line 40: @@
 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 Next GEM- Multiome – 3’ GEX and ATAC ===
+=== 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.
@@ Line 47: / Line 47: @@
 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 =

@@ Line 37: / Line 37: @@
 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 Next GEM 5’ – V(D)J ===
+=== 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.

← Older revision		Revision as of 13:39, 4 August 2023
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	=== 10X - Single cell - 3p ===		=== 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~~.	+	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.

← Older revision		Revision as of 18:54, 13 July 2023
Line 36:		Line 36:
	= Sequencing =		= 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.	+	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.