GSM2043599: villous_S2

Source name - placental villous tissue
Organism - Homo sapiens
Characteristics ercc spike-in pool: Mix_2
fetal sex: Female
gestational age (weeks): 40.86
tissue: placental villous tissue
Treatment protocol - Placenta samples were collected and dissected post-delivery and incubated in RNAlater solution at 4 degrees celcius for 24 hours before being stored at -80 degrees celcius.
Extracted molecule - total RNA
Extraction protocol - RNA was extracted from 16 placental samples using TRIzol following the manufacturer’s protocol. All samples were spiked with mix 1 or mix 2 of the 96 External RNA Controls Consortium (ERCC) ExFold RNA transcripts.
Ribosomal RNAs were depleted from samples using Ribo-Zero Gold. and sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA Sample Preparation kits. Sequencing was performed on the Illumina Hi-Seq 2500 using a 100bp paired-end protocol at the Australian Cancer Genomics Facility in Adelaide.

Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500

Data processing - Libraries were sequenced on the Illumina Hi-Seq 2500. Basecalls were performed with CASAVA version 1.8.
Sequence adapters were trimmed using AdapterRemoval with options --trimns, --minlength 20. Trimmed RNA-Seq reads were aligned to known UCSC hg19 genes and the hg19 genome using Bowtie 2 v2.1.0 and TopHat v2.0.9 with options --library-type=fr-firststrand --mate-inner-dist -20 --mate-std-dev 180. UCSC hg19 reference genome and transcriptome was obtained through Illumina iGenomes (https://support.illumina.com/sequencing/sequencing_software/igenome.html).
Aligned RNA-Seq reads were summarised using the summarizeOverlaps algorithm with the UCSC known genes hg19 GTF file using the the options overlapMode=``Union'', ignoreStrand=FALSE, singleEnd=FALSE, fragments=TRUE to generate a table of unique read counts per gene for each sample.
Genome_build: hg19
Supplementary_files_format_and_content: A count table of uniquely mapped read pairs overlapping genes.

样本来源名称（Source name） - 胎盘绒毛组织（placental villous tissue）
生物来源（Organism） - 智人（Homo sapiens）
样本特征（Characteristics）：
ERCC外参混合池（ercc spike-in pool）：Mix_2
胎儿性别（fetal sex）：女性（Female）
孕周（gestational age (weeks)）：40.86周
组织类型（tissue）：胎盘绒毛组织
处理方案（Treatment protocol）：胎盘样本于分娩后采集并解剖，置于4℃的RNAlater溶液中孵育24小时，随后转移至-80℃保存。
提取分子（Extracted molecule） - 总RNA（total RNA）
提取方案（Extraction protocol）：采用TRIzol试剂按照厂商说明书从16份胎盘样本中提取总RNA，所有样本均加入96种外部RNA对照联盟（External RNA Controls Consortium, ERCC）ExFold RNA转录本的Mix 1或Mix 2作为外参掺入。使用Ribo-Zero Gold试剂盒去除样本中的核糖体RNA，随后采用Illumina TruSeq Stranded Total RNA样本制备试剂盒构建测序文库。测序工作于阿德莱德的澳大利亚癌症基因组学设施完成，采用Illumina HiSeq 2500平台，以100bp双端测序策略进行测序。

文库构建策略（Library strategy）：RNA测序（RNA-Seq）
文库来源（Library source）：转录组（transcriptomic）
文库筛选方式（Library selection）：cDNA
测序仪器型号（Instrument model）：Illumina HiSeq 2500

数据处理流程（Data processing）：
- 采用Illumina HiSeq 2500平台完成文库测序。碱基识别工作采用CASAVA 1.8版本完成。使用AdapterRemoval工具并设置参数--trimns、--minlength 20修剪测序接头。将修剪后的RNA测序读段（reads）比对至UCSC hg19已知基因集及hg19参考基因组，使用Bowtie 2 v2.1.0和TopHat v2.0.9工具，参数设置为--library-type=fr-firststrand --mate-inner-dist -20 --mate-std-dev 180。UCSC hg19参考基因组及转录组通过Illumina iGenomes平台（https://support.illumina.com/sequencing/sequencing_software/igenome.html）获取。
- 使用summarizeOverlaps算法，结合UCSC已知基因hg19 GTF注释文件，设置参数overlapMode="Union", ignoreStrand=FALSE, singleEnd=FALSE, fragments=TRUE，对比对后的RNA测序读段进行计数汇总，生成每个样本各基因的唯一读段计数表。
基因组版本（Genome_build）：hg19
补充文件格式及内容（Supplementary_files_format_and_content）：包含与基因区域重叠的唯一比对读段对的计数表。