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#314 Genetics & Genomics Complete

RNA-Seq: HER2 KD vs scr in SK-BR-3 / HeLa / MCF7

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Tags

RNA-Seq HER2 DESeq2 transcriptomics breast-cancer

Custom ID

This template captures a bulk RNA-Seq differential expression study comparing HER2 siRNA knockdown to scrambled siRNA control across three breast cancer cell lines.

Goal: identify transcripts differentially expressed in response to HER2 knockdown, with three biological replicates per condition across SK-BR-3 (HER2 high), HeLa (HER2 low/neg), and MCF7 (HER2 low). 24 samples total: 3 cell lines × 2 conditions × 4 replicates (one extra to retain in case of QC failure).

Method overview: Total RNA is extracted from cell pellets (Qiagen RNeasy), RIN measured on Bioanalyzer (cutoff RIN > 8). Libraries are prepared with NEBNext Ultra II Directional RNA Library Prep Kit (polyA selection), 11 PCR cycles, dual-indexed. Library yield and fragment size are confirmed on the Bioanalyzer (target peak 270-340 bp). Pooled libraries are sequenced on Illumina NextSeq 2000 with a P2 100-cycle kit, paired-end 75, targeting ~25M reads per sample. Reads are trimmed (fastp), aligned to GRCh38 (STAR), quantified per gene (featureCounts), and differential expression run in DESeq2 with cell-line as a covariate.

Expected: HER2 (ERBB2) the top down-regulated transcript in KD condition (log2FC ~-2.5, padj < 1e-15). 200-500 DEGs at |log2FC| > 1 + padj < 0.05, enriched in ERBB signaling pathway gene sets.

Last saved: 34 seconds ago

12 fields loaded from RNA-Seq Cohort baseline (SOP v1.5)

Sample

Study design

select

2-group (case/ctrl), time course, multi-condition factorial

Sample group

text

siHER2 / siScr (per cell line)

Bio replicates / arm

number

4

RIN (mean)

number

9.4

Library

Library prep kit

text

NEBNext Ultra II Directional (polyA)

PCR cycles

number

11

Library yield (mean)

number

142ng/µL

Fragment size (peak)

number

312bp

Sequencing Run

Instrument

select

NextSeq 2000, NovaSeq 6000, MiSeq, NextSeq 550

Kit

text

P2 100-cycle, paired-end 75

Expected reads/sample

number

25million

Index strategy

select

dual-index, single-index, UMI-tagged

cohort_sample_sheet.csv

12 KB

library_QC_bioanalyzer.pdf

1.8 MB

deseq2_results.xlsx

4.2 MB

01

Total RNA extraction from cell pellets (Qiagen RNeasy)

24 samples · all RIN > 8

1:30

02

RIN QC on Bioanalyzer 2100 (Pico chip), QC reject any RIN < 8

All pass · mean RIN 9.4

1:00

03

NEBNext Ultra II directional library prep (polyA selection, 11 PCR cycles)

24 dual-indexed libraries

6:30

04

Library QC: Bioanalyzer + Qubit dsDNA HS quantification

Mean yield 142 ng/µL · peak 312 bp

1:30

05

Equimolar pooling: 4 nM each, 24-plex pool, store at -20°C

0:45

06

Denature + dilute pool, load onto NextSeq 2000 P2 100-cycle PE75 cartridge

Run started 2026-04-22 16:40

0:30

07

Sequencing run (Illumina NextSeq 2000, PE75, ~14 h)

Q30 > 90% · cluster density 220 K/mm²

14:00

08

BCL → FASTQ conversion, demultiplex, transfer to cluster

23-27 M reads / sample

1:00

09

Snakemake pipeline: fastp → STAR → featureCounts

92-96% uniquely mapped

3:30

10

DESeq2 DE analysis: ~siHER2 vs siScr + cell_line covariate

347 DEGs · HER2 top hit · padj 1.2e-18

0:45

	A	B	C	D	E
1	Gene	log2FC	padj	baseMean	Notes
2	ERBB2	-2.49	1.2e-18	14,820	HER2 — target of KD ✓
3	GRB7	-1.96	4.7e-12	3,240	ERBB2 amplicon neighbor
4	MIEN1	-1.74	8.3e-10	2,180	ERBB2 amplicon neighbor
5	PGAP3	-1.62	2.1e-09	1,985	ERBB2 amplicon neighbor
6	BCL2	-1.18	3.4e-06	4,420	Anti-apoptotic, ERBB-regulated
7	BIRC3	+1.42	5.7e-05	1,128	IAP family, pro-survival shift
8	CDKN1A	+1.27	1.2e-04	2,841	p21, cell-cycle arrest
9	...	...	...	...	347 DEGs total at padj<0.05

EXP-2026-0412 siRNA knockdown + RNA harvest (24 samples) D. Park Apr 12

EXP-2026-0310 qPCR HER2 KD validation (upstream check) Dr. S. Chen Mar 10

SOP-0511 RNA-Seq Cohort SOP v1.5 (extraction → DE) QA Team May 4

WORK-0073 Snakemake bulk-RNA-Seq workflow v2.4 Bioinf DB Apr 1

REF-0011 GRCh38 / GENCODE v45 reference + STAR index Refs DB Mar 28

NEBNext Ultra II Directional RNA Library Prep Kit

E7760S · Lot 10174831

In stock

NEBNext Multiplex Oligos for Illumina (dual)

E7600S · Lot 10174142

In stock

Bioanalyzer RNA 6000 Pico Chip

5067-1513 · Lot 1042

Low

Bioanalyzer DNA HS Chip

5067-4626 · Lot 0871

In stock

Illumina NextSeq 2000 P2 100-cycle kit

20046811 · Lot 20420314

In stock

AMPure XP beads

A63881 · Lot 21452

In stock

Total RNA aliquots (per cell line)

-80 °C · Freezer A-3 · Drawer 1 · Box RNA-2026-04

×24

Prepped indexed libraries (4 nM)

-20 °C · Freezer B-2 · Rack 1 · Box LIB-2026-04

×24

Pool (24-plex, ready to load)

-20 °C · Freezer B-2 · Rack 1 · Tube POOL-04

×1

Visibility

Park LabD. ParkC. Iverson

Can write

D. ParkC. IversonDr. K. Williams

Color

Stroke width

Eraser

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{
    "experiment": {
        "id": 314,
        "custom_id": "RNASEQ-HER2-2026-04",
        "title": "RNA-Seq: HER2 KD vs scr in SK-BR-3 / HeLa / MCF7",
        "template": "RNA-Seq",
        "template_version": "v3.2",
        "category": "Genetics & Genomics",
        "status": "Complete",
        "started_on": "2026-07-22",
        "tags": [
            "RNA-Seq",
            "HER2",
            "DESeq2",
            "transcriptomics",
            "breast-cancer"
        ]
    },
    "main_text": "This template captures a bulk RNA-Seq differential expression study comparing HER2 siRNA knockdown to scrambled siRNA control across three breast cancer cell lines.\nGoal: identify transcripts differentially expressed in response to HER2 knockdown, with three biological replicates per condition across SK-BR-3 (HER2 high), HeLa (HER2 low/neg), and MCF7 (HER2 low). 24 samples total: 3 cell lines × 2 conditions × 4 replicates (one extra to retain in case of QC failure).\nMethod overview: Total RNA is extracted from cell pellets (Qiagen RNeasy), RIN measured on Bioanalyzer (cutoff RIN > 8). Libraries are prepared with NEBNext Ultra II Directional RNA Library Prep Kit (polyA selection), 11 PCR cycles, dual-indexed. Library yield and fragment size are confirmed on the Bioanalyzer (target peak 270-340 bp). Pooled libraries are sequenced on Illumina NextSeq 2000 with a P2 100-cycle kit, paired-end 75, targeting ~25M reads per sample. Reads are trimmed (fastp), aligned to GRCh38 (STAR), quantified per gene (featureCounts), and differential expression run in DESeq2 with cell-line as a covariate.\nExpected: HER2 (ERBB2) the top down-regulated transcript in KD condition (log2FC ~-2.5, padj < 1e-15). 200-500 DEGs at |log2FC| > 1 + padj < 0.05, enriched in ERBB signaling pathway gene sets.",
    "extra_fields": {
        "Sample": {
            "Study design": {
                "type": "select",
                "value": "2-group (case/ctrl), time course, multi-condition factorial"
            },
            "Sample group": {
                "type": "text",
                "value": "siHER2 / siScr (per cell line)"
            },
            "Bio replicates / arm": {
                "type": "number",
                "value": "4"
            },
            "RIN (mean)": {
                "type": "number",
                "value": "9.4"
            }
        },
        "Library": {
            "Library prep kit": {
                "type": "text",
                "value": "NEBNext Ultra II Directional (polyA)"
            },
            "PCR cycles": {
                "type": "number",
                "value": "11"
            },
            "Library yield (mean)": {
                "type": "number",
                "value": "142 ng/µL"
            },
            "Fragment size (peak)": {
                "type": "number",
                "value": "312 bp"
            }
        },
        "Sequencing Run": {
            "Instrument": {
                "type": "select",
                "value": "NextSeq 2000, NovaSeq 6000, MiSeq, NextSeq 550"
            },
            "Kit": {
                "type": "text",
                "value": "P2 100-cycle, paired-end 75"
            },
            "Expected reads/sample": {
                "type": "number",
                "value": "25 million"
            },
            "Index strategy": {
                "type": "select",
                "value": "dual-index, single-index, UMI-tagged"
            }
        }
    },
    "attached_files": [
        {
            "filename": "cohort_sample_sheet.csv",
            "type": "csv",
            "size": "12 KB"
        },
        {
            "filename": "library_QC_bioanalyzer.pdf",
            "type": "pdf",
            "size": "1.8 MB"
        },
        {
            "filename": "deseq2_results.xlsx",
            "type": "xlsx",
            "size": "4.2 MB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Total RNA extraction from cell pellets (Qiagen RNeasy)",
            "duration": "1:30",
            "complete": true,
            "note": "24 samples · all RIN > 8"
        },
        {
            "n": 2,
            "description": "RIN QC on Bioanalyzer 2100 (Pico chip), QC reject any RIN < 8",
            "duration": "1:00",
            "complete": true,
            "note": "All pass · mean RIN 9.4"
        },
        {
            "n": 3,
            "description": "NEBNext Ultra II directional library prep (polyA selection, 11 PCR cycles)",
            "duration": "6:30",
            "complete": true,
            "note": "24 dual-indexed libraries"
        },
        {
            "n": 4,
            "description": "Library QC: Bioanalyzer + Qubit dsDNA HS quantification",
            "duration": "1:30",
            "complete": true,
            "note": "Mean yield 142 ng/µL · peak 312 bp"
        },
        {
            "n": 5,
            "description": "Equimolar pooling: 4 nM each, 24-plex pool, store at -20°C",
            "duration": "0:45",
            "complete": true
        },
        {
            "n": 6,
            "description": "Denature + dilute pool, load onto NextSeq 2000 P2 100-cycle PE75 cartridge",
            "duration": "0:30",
            "complete": true,
            "note": "Run started 2026-04-22 16:40"
        },
        {
            "n": 7,
            "description": "Sequencing run (Illumina NextSeq 2000, PE75, ~14 h)",
            "duration": "14:00",
            "complete": true,
            "note": "Q30 > 90% · cluster density 220 K/mm²"
        },
        {
            "n": 8,
            "description": "BCL → FASTQ conversion, demultiplex, transfer to cluster",
            "duration": "1:00",
            "complete": true,
            "note": "23-27 M reads / sample"
        },
        {
            "n": 9,
            "description": "Snakemake pipeline: fastp → STAR → featureCounts",
            "duration": "3:30",
            "complete": true,
            "note": "92-96% uniquely mapped"
        },
        {
            "n": 10,
            "description": "DESeq2 DE analysis: ~siHER2 vs siScr + cell_line covariate",
            "duration": "0:45",
            "complete": true,
            "note": "347 DEGs · HER2 top hit · padj 1.2e-18"
        }
    ],
    "spreadsheet": [
        [
            "Gene",
            "log2FC",
            "padj",
            "baseMean",
            "Notes"
        ],
        [
            "ERBB2",
            "-2.49",
            "1.2e-18",
            "14,820",
            "HER2 — target of KD ✓"
        ],
        [
            "GRB7",
            "-1.96",
            "4.7e-12",
            "3,240",
            "ERBB2 amplicon neighbor"
        ],
        [
            "MIEN1",
            "-1.74",
            "8.3e-10",
            "2,180",
            "ERBB2 amplicon neighbor"
        ],
        [
            "PGAP3",
            "-1.62",
            "2.1e-09",
            "1,985",
            "ERBB2 amplicon neighbor"
        ],
        [
            "BCL2",
            "-1.18",
            "3.4e-06",
            "4,420",
            "Anti-apoptotic, ERBB-regulated"
        ],
        [
            "BIRC3",
            "+1.42",
            "5.7e-05",
            "1,128",
            "IAP family, pro-survival shift"
        ],
        [
            "CDKN1A",
            "+1.27",
            "1.2e-04",
            "2,841",
            "p21, cell-cycle arrest"
        ],
        [
            "...",
            "...",
            "...",
            "...",
            "347 DEGs total at padj<0.05"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0412",
                "title": "siRNA knockdown + RNA harvest (24 samples)",
                "owner": "D. Park",
                "date": "Apr 12"
            },
            {
                "ref_id": "EXP-2026-0310",
                "title": "qPCR HER2 KD validation (upstream check)",
                "owner": "Dr. S. Chen",
                "date": "Mar 10"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0511",
                "title": "RNA-Seq Cohort SOP v1.5 (extraction → DE)",
                "owner": "QA Team",
                "date": "May 4"
            },
            {
                "ref_id": "WORK-0073",
                "title": "Snakemake bulk-RNA-Seq workflow v2.4",
                "owner": "Bioinf DB",
                "date": "Apr 1"
            },
            {
                "ref_id": "REF-0011",
                "title": "GRCh38 / GENCODE v45 reference + STAR index",
                "owner": "Refs DB",
                "date": "Mar 28"
            }
        ]
    },
    "compounds": [
        {
            "name": "NEBNext Ultra II Directional RNA Library Prep Kit",
            "catalog_lot": "E7760S · Lot 10174831",
            "stock_status": "In stock"
        },
        {
            "name": "NEBNext Multiplex Oligos for Illumina (dual)",
            "catalog_lot": "E7600S · Lot 10174142",
            "stock_status": "In stock"
        },
        {
            "name": "Bioanalyzer RNA 6000 Pico Chip",
            "catalog_lot": "5067-1513 · Lot 1042",
            "stock_status": "Low"
        },
        {
            "name": "Bioanalyzer DNA HS Chip",
            "catalog_lot": "5067-4626 · Lot 0871",
            "stock_status": "In stock"
        },
        {
            "name": "Illumina NextSeq 2000 P2 100-cycle kit",
            "catalog_lot": "20046811 · Lot 20420314",
            "stock_status": "In stock"
        },
        {
            "name": "AMPure XP beads",
            "catalog_lot": "A63881 · Lot 21452",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "Total RNA aliquots (per cell line)",
            "location": "-80 °C · Freezer A-3 · Drawer 1 · Box RNA-2026-04",
            "count": "×24"
        },
        {
            "name": "Prepped indexed libraries (4 nM)",
            "location": "-20 °C · Freezer B-2 · Rack 1 · Box LIB-2026-04",
            "count": "×24"
        },
        {
            "name": "Pool (24-plex, ready to load)",
            "location": "-20 °C · Freezer B-2 · Rack 1 · Tube POOL-04",
            "count": "×1"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Park Lab"
            },
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                "type": "Person",
                "name": "D. Park"
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                "name": "C. Iverson"
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                "name": "Dr. K. Williams"
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}