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Sample GSM7977654 Query DataSets for GSM7977654
Status Public on Dec 25, 2023
Title Central forebrain, ZT22 undesturbed, rep2 [ZT22-2]
Sample type SRA
 
Source name central forebrain
Organism Rattus norvegicus
Characteristics tissue: central forebrain
cell line: age 7-9 weeks
cell type: male
genotype: Long Evans
treatment: ZT22 undesturbed
Treatment protocol Thirty rats were randomly assigned to one of six groups (n=5/group) that were sampled every 4 h, beginning 2 h after light onset (i.e., ZT2, 6, 10, 14, 18, and 22). For the sleep deprivation (SD) study, twenty rats were randomly assigned to 6 h SD from ZT0–6, wherein rats were kept awake by an automated bedding stir bar (Pinnacle) at the bottom of a cylindrical cage. The bar was set to rotate for 4 s, randomly changing rotation direction and stopped for a random interval ranging from 10 to 30 s28,29. Following SD, rats (n=5/time point) were euthanized immediately (R0) or were returned to their home cage for 2 h (R2), 4 h (R4), or 8 h (R8) under red light without disruption before sampling. Five additional rats were euthanized at ZT8 as undisturbed, time-matched controls.
Growth protocol Male Long Evans rats (7-9 weeks old) were housed in pairs at 22 ± 2 °C on a 12:12 h light-dark cycle. The rats were acclimated to this light cycle for at least 10 days prior to tissue collection, with water and chow ad libitum. Cages were cleaned weekly (between 8 and 11 AM) unless the rats were being euthanized within 24 h.
Extracted molecule polyA RNA
Extraction protocol Rats were decapitated by guillotine under normal room light (ZT2–10) or under dim red light (ZT14–22). Following decapitation, forebrains were resected (Fig. 1A), frozen in 2-methylbutane suspended in dry ice, and then stored at -80 °C until homogenization for RNA extraction. Just before RNA isolation, forebrains were removed from -80 °C storage and placed on dry ice. Prior to use, a stainless-steel mortar and pestle were cleaned with RNase Zap (Thermo Fisher) and 70% ethanol. The mortar was then partially filled with liquid nitrogen before a forebrain was added, pulverized, and placed in a conical tube. Between each sample, the mortar and pestle were cleaned with 70% ethanol. A small aliquot of sample was removed for RNA isolation using Trizol Reagent (Invitrogen), according to the manufacturer’s instructions. Purified RNA was resuspended in water, and concentration and purity were measured with a Nanodrop spectrophotometer (Thermo Fisher).
WTTS-Seq libraries were prepared as described by Zhou et al. https://doi.org/10.1534/genetics.116.188508. Briefly, total RNA (2.5 µg) was incubated at 70 °C with 10X Fragmentation buffer (Invitrogen) for 3 min. The fragmentation reaction was halted by the addition of Stop Solution and incubation on ice for at least 2 min. Next, poly(A)+ RNA was purified from the fragmented total RNA with Dynabeads Oligo (dT)25 (Invitrogen), according to the manufacturer’s directions, and used for first-strand cDNA synthesis in a 20 µL reaction mixture. First, 1.0 µL of barcode primer (100 µM) and 1.0 µL of a common SMART primer (100 µM) were annealed to the poly(A)+ RNA template by heating to 65 °C for 5 min and incubating on ice for at least 2 min. Next, 4.0 µL of 5X First-strand buffer (Invitrogen), 1.0 µL of SuperScript III reverse transcriptase (Invitrogen), 1.0 µL of 0.1 M dithiothreitol, 2.5 µL of 10 mM dNTP, and 1.0 µL of RNase OUT (Invitrogen) were added to the mixture. First-strand cDNA was synthesized by incubating the mixture at 40 °C for 90 min in the presence of library-specific adaptors. Synthesis was terminated by heating the mixture at 70 °C for 15 min. RNases I (100 U/µL; Invitrogen) and H (2 U/µL; Invitrogen) were subsequently added and incubated with the mixture at 37 °C for 30 min to hydrolyze the remaining single-stranded RNA molecules and ensure that only single-stranded cDNA remained. RNase activities were terminated by heating the samples at 70 °C for 20 min. Following purification with solid-phase reversible immobilization (SPRI) beads, second-strand cDNA was synthesized from first-strand cDNA by asymmetric PCR. In addition to the cDNA, the 50 µL PCR reaction contained 1.0 µL of Phusion Hi-Fidelity DNA polymerase, 10.0 µL of 5X HF buffer, 1.0 µL of 0.4 µM barcode primer, 1.0 µL of 0.8 µM common primer, 1.0 µL of 10 mM dNTP, and nuclease-free water. The PCR reaction was carried out by heating at 95 °C for 30 sec, followed by 20 cycles of 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s, with a final elongation step at 72 °C for 10 min. SPRI beads were used to purify and select 200 – 500 bp fragments from the final library.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Ion Torrent PGM
 
Description WTTS-seq targets polyA-adjacent sequence
Raw_counts_WTTS_Gerstner_03202022
Raw_counts_no_mito_WTTS_Gerstner_03202022
meta2d_12h_no_mito_WTTS
meta2d_24h_no_mito_WTTS
Data processing *library strategy: WTTS-seq (whole transcriptome termini site sequencing) targeting alternative polyA sites
Raw read processing: Raw data were obtained from 55 samples and stored in FASTQ format. We filtered raw reads with the FASTQ quality filter in the FASTX Toolkit (v0.0.13), allowing for a minimum score of ≥10 for ≥50% of bases (http://hannonlab.cshl.edu/fastx_toolkit/). We trimmed T nucleotides or T-rich sequences located at the 5’ ends of the reads using Perl scripts, as described in a previous study30. Trimmed reads of at least 16 bp in length were kept for further analysis.
Read mapping and poly(A) site clustering: For each data set, we aligned the processed reads to the Rattus norvegicus genome (mRatBN7.2/rn7) using the torrent mapping program (TMAP, v3.4.1; http://github.com/iontorrent/tmap) with the unique best hits parameter (-a 0). We then extracted raw PASs supported by the uniquely mapped reads from SAM files and obtained a polyadenylation tag (PAT) file using an in-house script. After that, we merged all the PAT files to determine the final PASs for all samples. PASs within 25 nucleotides of one another were grouped into one polyadenylation site cluster (PAC) using GetPolyaSiteCluster31. PACs with few reads were rejected according to different criteria depending on library size. For libraries that had less than 1.7M reads, clusters were retained only if a group of 5 biological replicates had at least 3 samples with ≥3 reads. For libraries with more than 1.7M reads, at least 3 samples with ≥4 reads were required.
Gene annotation and usage of poly(A) sites: We annotated all the final PACs for PAS_ID, gene symbol, functional region, and other factors, as indicated, using Cuffcompare (v2.2.1)32, Perl scripts, and annotation file (GCF_000001895.5_Rnor_6.0_genomic.gtf; https://ftp.ncbi.nlm.nih.gov/). Clusters that mapped to mitochondrial genes were removed, then the number of PAS-covered reads was normalized33 to the total number of covered reads within each library and rescaled by a factor of 107.
Circadian/ultradian PAS discovery: Based on input, unnormalized PAS read counts, rhythmic patterns were identified using the MetaCycle34 R package meta2d, which synthesizes the results of three cycle analysis algorithms (ARSER, JTK_Cycle, and Lomb-Scargle). The analysis was run 5 times with different replicates inserted into each of the appropriate ZT time slots, and the average p-value, FDR, phase, and other statistics were calculated. Only the highly corroborated PASs that were significant (p<0.05) in all 5 trials were used for all analyses.
Differential expression analysis of sleep deprivation/recovery: To evaluate the expression of PASs in sleep homeostasis experiments, PAS counts from rats recovering from 6 h SD were contrasted with time-matched controls (R0 vs ZT6, R2 vs ZT8, R4 vs ZT10, R8 vs ZT14). We removed high variation from the first principal component systematically, resulting in improved variance estimates for low read counts. Prcomp (in R) was used to perform principal component analysis (PCA) and find eigenvectors by way of singular value decomposition. DESeq-2 with "Apeglm" Shrinkage36 and the Wald Test were used to generate test statistics in R software. The FDRtool was used to determine the Local FDR
Assembly: Rattus norvegicus genome build mRatBN7.2/rn7
Supplementary files format and content: the details in the processed_data_readme.txt
 
Submission date Dec 15, 2023
Last update date Dec 25, 2023
Contact name Jason Robert Gerstner
E-mail(s) j.gerstner@wsu.edu
Organization name Washington State Univ. Spokane
Department Translational Medicine and Physiology
Lab Gerstner
Street address 412 E Spokane Falls BLVD
City Spokane
State/province WA
ZIP/Postal code 99201
Country USA
 
Platform ID GPL20670
Series (1)
GSE250324 Identification of sleep and circadian alternative polyadenylation sites associated with APA-linked human brain disorders
Relations
BioSample SAMN38859097
SRA SRX22906316

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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