Resources

Services List

Extractions (DNA or RNA)

Sample Type	Input Requirements*	Price
Cells	0.5-10 million cells	$32.00
Tissue	5-30mg tissue	$32.00
Paraffin-embedded (FFPE) Tissue	Please provide three 5mm sections	$47.00
Saliva	Sample provided in a DNAGENOTEK Saliva-based collection tube	$47.00
Blood/Serum/Plasma/Buffy Coat	Sample provided in BD Vacutainer Blood Collection tube(www.bd.com), Paxgene tube, or Tempus tube Note: please follow manufacturer protocol/recommendations for each tube type	$47.00

*Input requirements are recommended. If your sample amount is limited, contact us to discuss options.

Library Preparation - Input Requirements

RNA Library Preparation	Input Quantity Range	RNA Integrity (RIN) Score	Min Concentration
Illumina Stranded Total Ligation (Ribo-Zero Plus)	10-1000 ng	RIN > 2	1 ng/µl
Illumina Stranded mRNA (PolyA+)	25-1000 ng	RIN ≥ 8	1 ng/µl
SMARTer TCR Profiling (Human/mouse)	10-250 ng	RIN > 7	1 ng/µl
SMART-Seq Stranded Total RNA (Ultra Low Input)	1-10 ng	RIN > 2	0.13 ng/µl
SMART-Seq HT Ultra Low Input	1-10 ng	RIN ≥ 8	0.2 ng/µl
QIAseq small RNA	1-500 ng	RIN ≥ 8	0.2 ng/µl
DNA Library Preparation
Nextera DNA XT (low input)	1 ng cDNA		0.2 ng/µl
Illumina DNA Prep (Nextera FLEX)	1-500 ng cDNA		0.2 ng/µl
Illumina DNA PCR-Free Prep (WGS)	25-300 ng DNA		1 ng/µl
ChIP-seq (Library prep only)	5-10 ng CHIP DNA		0.1 ng/µl
ATAC-Seq	10k-50k cells

Library Generation

Illumina Stranded Total Ligation (Ribo-Zero Plus)
Total RNA input is depleted using enzymatic depletion method that removes abundant transcripts from total RNA. Random primers initiate first and second strand cDNA synthesis. Adenylation of 3' ends are followed by adapter ligation and library amplification with indexing. 

This method is used when you want to characterize long non-coding RNA (lncRNA) as well as mRNA. It is compatible with degraded RNA as there is no poly-A enrichment.

Illumina Stranded mRNA (PolyA+)
Total RNA input is enriched for mRNA molecules containing polyA tails using Oligo(dT) magnetic beads. The purified mRNA is fragmented, and random primers initiate first strand and second strand cDNA synthesis. Adenylation of 3' ends are followed by adapter ligation and library amplification with indexing. 

This method is used in transcriptome or gene expression studies.

SMARTer TCR Profiling (Human/mouse)
Used to analyze T-cell receptor (TCR) diversity from total (bulk) RNA samples and whole cells. cDNA is synthesized using TCR dT primers. Two rounds of PCR are performed to amplify cDNA sequences. In the second PCR, forward and reverse primers are added including adapter and index sequences.

SMART-Seq Stranded for Total RNAseq (Ultra Low Input)
Reverse transcription with random primers and template switching generates first strand cDNA. cDNA is amplified using the template switching adapter and a sequence added to the random primers. Ribosomal cDNA is cleaved and the uncleaved fragments are amplified with indexed primers. This method is used with low quality and quantity RNA samples.

SMART-Seq mRNA HT (Ultra Low Input) – for flow sorted cells
SMART-Seq HT ultra-Low input RNA Kit – prepare ds-cDNA  

Reverse transcription with oligo(dT) primers and template switching oligo generates first strand cDNA. cDNA is amplified using the template switching adapter and a sequence added to the polyT primer. 

This kit is used for very low input RNA and/or direct flow sorted samples.

Illumina Nextera XT
Tagmentation with transposase fragments DNA and incorporates shortened Illumina adapters. Indexes are added via index PCR.

Illumina DNA Prep (previously named: Nextera FLEX)
Tagmentation using enzymatic reaction to fragment DNA and add adapter sequences. Indexes are added via index PCR.

Illumina DNA PCR-free
Tagmentation using enzymatic reaction to fragment DNA and add adapter sequences. Indexes are added via direct ligation. Used for whole genome sequencing (WGS).

Lexogen 3'-Quantseq
QuantSeq kits enable cost-efficient sequencing by counting (differential gene expression only). These kits are an alternative to standard Total RNA-Seq from highly degraded low-input samples. Library is generated from 3'-ends only. Not recommended for lncRNA and isotype variants.

QIAseq small RNA
Pre-adenylated adapter is ligated to the 3' end of small RNAs followed by 5' ligation of an RNA adapter. cDNA synthesis is completed using a reverse transcription (RT) primer targeted to the 3' pre-adenylated adapter. Unique molecular indices (UMIs) are integrated into the RT primer to assign a unique identifier to each miRNA molecule. Indexes are added via index PCR.

ChIP-seq (Library prep only)
The goal of this protocol is to add adapter sequences onto the ends of ChIP DNA to generate indexed libraries.

ATAC-Seq/Omni-ATAC-seq
Assay for transposase-accessible chromatin sequencing to identify regions of open chromatin. In the tagmentation process, Tn5 transposase fragments open genomic DNA and inserts sequencing primers into chromatin open regions. Data analysis from sequencing provides understanding of gene regulation.

Illumina Sequencing

MiSeq Flow Cells	Number of Reads	Cycles	Typical Run Type	Price
Micro	4 million	300	2x150bp	$620
V2	13 million	50	1x50bp	$1,180
		300	2x150bp	$1,460
V3	25 million	150	2x75bp	$1,280
		600	2x300bp	$2,050
NextSeq 2000 Flow Cells	Number of Reads	Cycles	Typical Run Type	Price
P1	100 million	100	2x58bp	$1,300
		600	2x300bp
P2	400 million	100	2x58bp	$1,900
		200	2x100bp	$3,450
		300	2x150bp	$4,480
P3	1.2 billion	50	1x50bp, 2x35bp	$3,100
		100	2x58bp	$4,000
		200	2x100bp	$5,600
		300	2x150bp	$7,350

Single Cell Technologies

3' Gene Expression
Gene expression of up to 10,000 cells per sample. Multiplexing options include cell hashing with antibody labelling or CellPlex lipid tags.

5' Gene Expression and Immune Profiling
Gene expression, TCR and BCR profiling of up to 10,000 cells per sample. Multiplexing available with antibody-based cell hashing.

Gene Expression Flex
A probe-based assay that can be used to profile fixed tissues and cells (including cells isolated from FFPE). Tissues and cells can be stored up to 6 months at -80C. Multiplexing available for up to 16 samples. Compatible with human and mouse.

CITE-Seq
Barcoded antibodies bind to surface markers on individual cells. The barcode marker sequence carries over through the library preparation, permitting quantitative assessment of surface antibody binding to cells along with their transcriptome information. Hundreds of surface antigens can be assayed simultaneously.

Multiome ATAC and Gene Expression
Provides simultaneous detection of mRNA and chromatin accessibility from the same cell.

For more information, please visit https://singlecell.pitt.edu/

Spatial Transcriptomics

10X Visium Spatial Gene Expression

For additional information: https://www.10xgenomics.com/products/spatial-gene-expression 

Sample Type	Tissue Size	Samples per Slide	Staining Options	Cost (Sequencing not included)
Fresh Frozen	6.5mm x 6.5mm	4	H&E, Immunofluorescence (4 colors max)	$6,200 + $1,200 for tissue permeabilization (required)
CytAssist Fresh Frozen	6.5mm x 6.5mm	4	H&E only	$8,400
CytAssist Fresh Frozen	11mm x 11mm	2	H&E only	$8,600
CytAssist FFPE	6.5mm x 6.5mm	4	H&E, Immunofluorescence (4 colors max)	$8,400
CytAssist FFPE	11mm x 11mm	2	H&E, Immunofluorescence (4 colors max)	$8,600

Curio Biosciences Slide Seeker

For additional information: https://curiobioscience.com/ 

Sample Type	Tissue Size	Samples per Slide	Staining Options	Cost
Fresh Frozen	3mm x 3mm	1	None for transcriptome section – serial sections for staining recommended	Please contact
Fresh Frozen	10mm x 10mm	1	None for transcriptome section – serial sections for staining recommended	Please contact

Other Resources

Preparing Samples for Submission 

DNA/RNA Extraction

Sample Type	Sample Submission
Cells	After harvesting cells, do one of the following: 1. Pellet cells, snap freeze in liquid nitrogen and store at -90C to -65C. Deliver samples on dry ice. 2. Pellet cells, remove supernatant, store dry cell pellet at -80C. Deliver samples on dry ice. 3. Lyse cells in Buffer RLT (Contact HSSC for Buffer information). Deliver samples on wet ice.
Tissue / Paraffin-embedded Tissue (FFPE)	Deliver tissue on dry ice.
Saliva	Deliver at room temperature.
Blood, Serum, Plasma, Buffy Coat	Frozen samples: deliver on dry ice. Not frozen: deliver on wet ice.

NGS Samples for Library Preparation

Sample Type	Storage Conditions
RNA	Dry ice
DNA	Wet ice
Library	Wet ice or directly in freezer
Pooled Library	Wet ice or directly in freezer

Prepared Libraries

• Before making your libraries, please make sure your barcodes are compatible with Illumina Standards
• Minimum concentration: 2 nM (measured by fluorometry)
• Minimum volume: 15-20 μl

Single Cell Core

• Single cell samples should be brought to us as a cell suspension in PBS containing 0.04% BSA. Nuclei for Multiome processing should be brought in Nuclei Resuspension Buffer with RNAse Inhibitor. Fixed Cells for the Flex Assay should be prepared in long term storage buffer and delivered on dry ice.

• Cells should be as concentrated as possible. Samples run individually should be ~1 million live cells per mL. Samples intended for cell hashing should be ~3.5 million live cells per mL. Nuclei for Multiome processing should be 3.5 million nuclei per mL.

• Cell loading recommendations are 500-10,000 cells per well or up to 30,000 cells per well for hashed samples. The doublet rate and chances of clogging the chip increase with higher cell loading.

• If your sample dissociation protocol contains Tween-20 or EDTA, you will need to ensure that this is washed out of your sample prior to submission as this can prevent emulsification.

• Cells should be filtered through at least a 70 micron filter (30-40 micron recommended). Cells should be smaller than 54 microns (the channel size of the Chromium chip).

Drop-off Locations and Instructions

HSSC@CHP

1. Samples must be labeled with PI name, contact name, email and phone number.
2. Call (412-692-3555) or email us within 2 hours to let us know you have dropped off samples so we may retrieve them.
3. Send the sample submission form with all information. Samples cannot be processed without a proper sample submission form.

We are located in the Rangos Research Bldg, on the Children's Hospital Campus in Lawrenceville. There is a drop-off freezer just inside the sliding doors at the entrance to our building.

If you are driving, you can pull into the driveway for the Mid-Campus garage on 44th St. and follow past the Plaza Building/Ronald McDonald House to the front entrance to drop-off. There is a small area to turn around in or you can park in the Mid-campus garage (pay-parking for visitors and patient families).

Single Cell Core

The single cell core is located in S765 in the South BST. Our lab phone number is 412-383-5223.

Research Support

Facilities and Other Resources description for Grant applications:
Laboratory:

The Health Sciences Sequencing Core (HSSC) at Children’s Hospital of Pittsburgh is housed in the Richard King Mellon Institute for Pediatric Research, which is located on the 8th floor of the new Rangos Research Building as part of the Children’s Hospital of Pittsburgh of UPMC. The RK Mellon Institute for Pediatric Research is a multidisciplinary research institute comprising of physician scientists in the fields of Immunology, Developmental Biology, and Neurology. The John G. Rangos Research Center is a nine story, 144,000 square foot building located on the newly constructed, environmentally “green” campus of Children’s Hospital of Pittsburgh of UPMC. The total area of the RK Mellon Institute for Pediatric Research laboratories and facilities is around 10,000 square feet. Our facility represents around 500 square feet and offers next generation sequencing services to all University of Pittsburgh Researchers.

Next Generation Sequencing: The facility operates Illumina NextSeq 2000 & 500 instruments and an Illumina DRAGEN server on the NextSeq 2000 that can generate up to 330Gb of sequence data per flow cell. Instrument capacity is therefore readily available for large scale sequencing projects using sequencing by synthesis technology. Instruments can be operated in medium or high output to facilitate projects of any scale. In addition, the facility operates an Illumina MiSeq instrument for targeted resequencing, small genome, metagenomic and microbiome studies, as well as QC assessment. Together these platforms provide for a broad range of applications from targeted resequencing to genomic scale sequencing capability. A Fragment Analyzer (Advanced Analytical Inc.), a Qubit Fluorometer (Thermo Fisher) and a 4150 TapeStation (Agilent) are used for library QC and quantification. For Spatial Transcriptomics, the HSSC has a dedicated EVOS M7000 Imaging System equipped with 4x, 10x, 20x objectives, and GFP, DAPI, CY5, RFP light cubes for IF imaging workflows.

Major Equipment:

Facility dedicated equipment includes: one illumina NextSeq 2000 high throughput sequencer, one illumina NextSeq 500 high throughput sequencer, one illumina MiSeq sequencer, three Forma Freezers (-80°C; Upright); two Forma Freezers (-20°C; Upright); Agilent 4150 TapeSation; Advanced Analytical Inc. 5300 Fragment Analyzer; Invitrogen Qubit 4.0; Invitrogen Qubit FLEX; NanoDrop 2000 UV-Vis Spectrophotometer; Bio-Rad CFX96 Touch Real-Time PCR Detection System; two C1000 Touch Thermal Cyclers, two T100 Touch Thermal Cyclers, Liquid Nitrogen Storage Container DOT Scientific and multiple high-speed and ultra-centrifuges, EVOS M7000 Imaging System

Publication Acknowledgment:
Please acknowledge work from our core in all publications by including the following statement: This project utilized the services of the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh, for [name(s) of relevant service(s)].

Sample Submission Forms

• HSSC Sample Submission Form

• HSSC Sequencing Only Form

• Single Cell Core Sample Submission Form

• HTGC Project Inquiry Form

• HTGC Sample Submission Form

User (Service) Agreement

For providing services outside of Pitt, please click here to download the service agreement.