根据您提供的最新GEO提交流程和Excel模板，您需要将数据分为原始数据（FASTQ）和处理后数据（表达矩阵），并严格按照模板的Sheet结构进行填写。

为了确保您能直接复制粘贴到Excel模板中，我为您整理了Metadata Tab和MD5 Checksums Tab的所有必填内容。

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⚠️ 提交前的重要准备

1. 准备处理后数据（Processed Data）：GEO强制要求上传表达矩阵。请将您的18个样本的基因表达量（Raw counts 或 DESeq2 rlog normalized counts）合并为一个制表符分隔的文本文件，例如命名为 RNAseq_counts_matrix.txt。行名为基因Locus tag（如 H0N29_08675），列名为18个样本名（如 wt_r1, wt.abx_r2 等）。
2. 确认文件名：以下表格中的 raw file 名称已完全匹配您提供的 cp 命令中的目标文件名。请确保您实际上传的文件名与表格中完全一致（区分大小写）。

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第一部分：填写 Metadata Tab

请在Excel模板的 Metadata 工作表中，找到对应的区块（STUDY, SAMPLES, PROTOCOLS, PAIRED-END EXPERIMENTS），将以下内容复制粘贴到第B列（即Value列，不要修改A列的字段名）。

1. STUDY 区块

字段名 (Column A)	填写内容 (复制到 Column B)
*title	Transcriptomic responses of Acinetobacter baumannii ATCC19606 efflux pump mutants under chloramphenicol stress
*summary (abstract)	Efflux pumps play a key role in antibiotic resistance in Acinetobacter baumannii. Here, we investigated the transcriptomic responses of A. baumannii ATCC19606 mutants lacking the efflux pumps AdeAB, AdeIJ, or CraA under chloramphenicol stress. RNA-seq was performed on wild-type and knockout strains grown in LB medium with or without sub-MIC chloramphenicol. The data reveal strain-specific metabolic remodeling and highlight CraA as a major determinant of chloramphenicol resistance.
*experimental design	RNA-seq profiling of wild-type Acinetobacter baumannii ATCC19606 and its isogenic efflux pump knockout mutants (ΔadeAB, ΔadeIJ, and ΔcraA). Samples were cultured in LB medium under two conditions: untreated (control) and treated with sub-inhibitory concentration of chloramphenicol. Biological replicates (2 to 3 per condition) were sequenced using Illumina NovaSeq 6000 (paired-end, 150 bp).
contributor	Wuen Ee,Foong
contributor	Xinxin,Xiang
contributor	Wenjun,He
contributor	Xuan,Yan
contributor	Jiabin,Huang
contributor	Klaas M.,Pos
contributor	Heng-Keat,Tam
supplementary file	RNAseq_counts_matrix.txt

2. SAMPLES 区块 (共18行)

提示：在Excel中，将以下表格内容直接复制，然后选中SAMPLES区块的第一行数据单元格（即 `library name` 下方的单元格），右键“粘贴”即可。*

*library name	*title	*library strategy	*organism	strain	genotype	treatment	*molecule	*single or paired-end	*instrument model	description	processed data file	*raw file	raw file
wt_r1	WT, untreated, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt_r1	RNAseq_counts_matrix.txt	wt_r1_R1.fq.gz	wt_r1_R2.fq.gz
wt_r2	WT, untreated, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt_r2	RNAseq_counts_matrix.txt	wt_r2_R1.fq.gz	wt_r2_R2.fq.gz
wt_r3	WT, untreated, rep3	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt_r3	RNAseq_counts_matrix.txt	wt_r3_R1.fq.gz	wt_r3_R2.fq.gz
wt.abx_r1	WT, chloramphenicol, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt.abx_r1	RNAseq_counts_matrix.txt	wt.abx_r1_R1.fq.gz	wt.abx_r1_R2.fq.gz
wt.abx_r2	WT, chloramphenicol, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt.abx_r2	RNAseq_counts_matrix.txt	wt.abx_r2_R1.fq.gz	wt.abx_r2_R2.fq.gz
wt.abx_r3	WT, chloramphenicol, rep3	RNA-seq	Acinetobacter baumannii	ATCC 19606	Wild-type	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: wt.abx_r3	RNAseq_counts_matrix.txt	wt.abx_r3_R1.fq.gz	wt.abx_r3_R2.fq.gz
adeAB_r1	ΔadeAB, untreated, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeAB	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeAB_r1	RNAseq_counts_matrix.txt	adeAB_r1_R1.fq.gz	adeAB_r1_R2.fq.gz
adeAB_r2	ΔadeAB, untreated, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeAB	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeAB_r2	RNAseq_counts_matrix.txt	adeAB_r2_R1.fq.gz	adeAB_r2_R2.fq.gz
adeAB.abx_r1	ΔadeAB, chloramphenicol, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeAB	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeAB.abx_r1	RNAseq_counts_matrix.txt	adeAB.abx_r1_R1.fq.gz	adeAB.abx_r1_R2.fq.gz
adeAB.abx_r2	ΔadeAB, chloramphenicol, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeAB	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeAB.abx_r2	RNAseq_counts_matrix.txt	adeAB.abx_r2_R1.fq.gz	adeAB.abx_r2_R2.fq.gz
adeAB.abx_r3	ΔadeAB, chloramphenicol, rep3	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeAB	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeAB.abx_r3	RNAseq_counts_matrix.txt	adeAB.abx_r3_R1.fq.gz	adeAB.abx_r3_R2.fq.gz
adeIJ_r1	ΔadeIJ, untreated, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeIJ	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeIJ_r1	RNAseq_counts_matrix.txt	adeIJ_r1_R1.fq.gz	adeIJ_r1_R2.fq.gz
adeIJ_r2	ΔadeIJ, untreated, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeIJ	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeIJ_r2	RNAseq_counts_matrix.txt	adeIJ_r2_R1.fq.gz	adeIJ_r2_R2.fq.gz
adeIJ.abx_r1	ΔadeIJ, chloramphenicol, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeIJ	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeIJ.abx_r1	RNAseq_counts_matrix.txt	adeIJ.abx_r1_R1.fq.gz	adeIJ.abx_r1_R2.fq.gz
adeIJ.abx_r2	ΔadeIJ, chloramphenicol, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔadeIJ	Chloramphenicol	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: adeIJ.abx_r2	RNAseq_counts_matrix.txt	adeIJ.abx_r2_R1.fq.gz	adeIJ.abx_r2_R2.fq.gz
craA_r1	ΔcraA, untreated, rep1	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔcraA	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: craA_r1	RNAseq_counts_matrix.txt	craA_r1_R1.fq.gz	craA_r1_R2.fq.gz
craA_r2	ΔcraA, untreated, rep2	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔcraA	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: craA_r2	RNAseq_counts_matrix.txt	craA_r2_R1.fq.gz	craA_r2_R2.fq.gz
craA_r3	ΔcraA, untreated, rep3	RNA-seq	Acinetobacter baumannii	ATCC 19606	ΔcraA	Untreated	total RNA	paired-end	Illumina NovaSeq 6000	Column name in RNAseq_counts_matrix.txt: craA_r3	RNAseq_counts_matrix.txt	craA_r3_R1.fq.gz	craA_r3_R2.fq.gz

3. PROTOCOLS 区块

字段名 (Column A)	填写内容 (复制到 Column B)
growth protocol	Strains were cultured in sterile LB. Overnight cultures were inoculated into 50 mL of LB supplemented with or without sub-inhibitory concentration of chloramphenicol to an initial OD600 of 0.05, and incubated at 37°C with 130 rpm to an OD600 of 0.5-0.7.
*extract protocol	Cell suspensions were treated with RNAprotect Bacteria Reagent (Qiagen). Total RNA was extracted using RNeasy Mini Kit (Qiagen) with on-column DNA digestion and Turbo DNase treatment, followed by purification with RNeasy MinElute Cleanup kit.
*library construction protocol	Ribosomal RNA was removed using the Ribo-Zero kit. First-strand cDNA was synthesized using random hexamer primers, followed by second-strand synthesis, terminal repair, A-tailing, adapter ligation, size selection, and PCR enrichment.
*data processing step	Reads were processed using the nf-core/rnaseq v1.3dev pipeline.
data processing step	Quality trimming was performed using Trimmomatic v0.39.
data processing step	Reads were aligned to the reference genome CP059040 using HISAT2 v2.1.0.
data processing step	Gene expression levels were quantified using featureCounts.
data processing step	Raw counts were normalized using the regularized log transformation (rlog) from DESeq2.
*genome build/assembly	CP059040
*processed data files format and content	Tab-delimited text file containing raw counts and normalized (rlog) expression values for all 18 samples. Rows are genes (Locus tags), columns are samples.

4. PAIRED-END EXPERIMENTS 区块 (共36行)

提示：将以下两列内容复制，粘贴到 file name 1 和 file name 2 下方的单元格中。

file name 1	file name 2
wt_r1_R1.fq.gz	wt_r1_R2.fq.gz
wt_r2_R1.fq.gz	wt_r2_R2.fq.gz
wt_r3_R1.fq.gz	wt_r3_R2.fq.gz
wt.abx_r1_R1.fq.gz	wt.abx_r1_R2.fq.gz
wt.abx_r2_R1.fq.gz	wt.abx_r2_R2.fq.gz
wt.abx_r3_R1.fq.gz	wt.abx_r3_R2.fq.gz
adeAB_r1_R1.fq.gz	adeAB_r1_R2.fq.gz
adeAB_r2_R1.fq.gz	adeAB_r2_R2.fq.gz
adeAB.abx_r1_R1.fq.gz	adeAB.abx_r1_R2.fq.gz
adeAB.abx_r2_R1.fq.gz	adeAB.abx_r2_R2.fq.gz
adeAB.abx_r3_R1.fq.gz	adeAB.abx_r3_R2.fq.gz
adeIJ_r1_R1.fq.gz	adeIJ_r1_R2.fq.gz
adeIJ_r2_R1.fq.gz	adeIJ_r2_R2.fq.gz
adeIJ.abx_r1_R1.fq.gz	adeIJ.abx_r1_R2.fq.gz
adeIJ.abx_r2_R1.fq.gz	adeIJ.abx_r2_R2.fq.gz
craA_r1_R1.fq.gz	craA_r1_R2.fq.gz
craA_r2_R1.fq.gz	craA_r2_R2.fq.gz
craA_r3_R1.fq.gz	craA_r3_R2.fq.gz

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第二部分：填写 MD5 Checksums Tab (可选但强烈推荐)

GEO使用MD5校验和来验证文件在FTP上传过程中是否损坏。建议您在本地生成MD5值并填入此Sheet。

如何生成MD5值：

• Linux / macOS: 打开终端，进入文件所在目录，运行： md5sum *.fq.gz *.txt > md5_checksums.txt (Linux) 或 md5 *.fq.gz *.txt (macOS)
• Windows: 打开CMD，运行： certutil -hashfile wt_r1_R1.fq.gz MD5 (对每个文件运行)

将生成的哈希值填入 MD5 Checksums 工作表的对应列中：	file name (Raw files)	file checksum	file name (Processed)	file checksum
wt_r1_R1.fq.gz	(填入MD5值)	RNAseq_counts_matrix.txt	(填入MD5值)
wt_r1_R2.fq.gz	(填入MD5值)
…	…

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第三部分：实操上传步骤 (对应您列出的 Step 6 – Step 8)

1. 连接FTP：使用 FileZilla 或命令行 FTP 连接到 GEO 提供的服务器（通常是 ftp-private.ncbi.nlm.nih.gov），使用您的 NCBI 用户名和密码登录。
2. 创建文件夹：进入您的个人目录 /uploads/yourusername_abcd1234/，创建一个新文件夹，例如命名为 GEO_submission_Foong_2026。
3. 上传文件：
   • 将 36个 .fq.gz 文件 和 1个 RNAseq_counts_matrix.txt 文件 全部拖入该文件夹中。
   • ⚠️ 注意：不要将填好的 Excel 模板上传到 FTP！Excel 模板是通过网页提交的。
4. 网页提交 Metadata：
   • 登录 NCBI GEO Submission Portal。
   • 选择 “Submit Metadata”。
   • 上传您填好的 seq_template_Foong_2021_RNAseq.xlsx 文件。
5. 设置 Release Date：在提交系统中，您可以将数据的公开日期（Release Date）设置为论文正式发表的日期。在此之前，数据是保密的，但系统会生成一个 Reviewer Link，您可以将此链接附在论文的 Data Availability 声明中，供审稿人查看数据。

💡 最终核对清单 (Checklist)

• Excel 中的 RNAseq_counts_matrix.txt 文件名是否与您实际准备上传的矩阵文件名完全一致？（如果不一致，请在Excel中全局替换）。
• 表达矩阵的列名是否与 SAMPLES 区块中的 *library name（如 wt_r1, adeAB.abx_r2）完全一致？
• 所有的 .fq.gz 文件是否已经打包并准备通过 FTP 上传？