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Sample GSM7453782

Query DataSets for GSM7453782

Status

Public on Aug 25, 2023

Title

AF_CPN_4600

Sample type

SRA

Source name

sorted cortical neurons

Organism

Mus musculus

Characteristics

input: 4600 cells
fragmentation: Alkaline Fragmentation (pH=9.2, 37 min at 95 deg C)
Stage: P3
cell type: Callosal Projection Neurons

Treatment protocol

CPN: We performed electroporation of fluorescent constructs using CD-1 background animals at E14.5 or E15.5. Briefly, we exposed both uterine horns, and injected a solution of 5 ug/uL DNA + 0.1% FastGreen in 1X TE containing 5ug/uL plasmid encoding myristolated-TdTomato into one lateral ventricle in approximately ½ of embryos. We then electroporated per injected animal with 5 pulses of 500 ms each, 34 mV. We screened pups for red fluorescence in the cortex at P1 under an epifluorescent dissection scope. SCPN: We labeled SCPN by ultrasound-guided injection into the cerebral peduncle at P1 of B6SJL x CD-1 pups bred in-house. Briefly, we anaesthetized the pups by placing them on ice for 3 minutes, then securing to a solid surface. We then visualized the cerebral peduncle and placement of the injection needle via intracranial ultrasound. We injected Alexa 555-Cholera Toxin B (CTB) into the cerebral peduncle, injecting 4 sites (at slightly different dorsal, ventral, medial and lateral sites to ensure coverage of the peduncle, with five 5 nl pulses per site), all into the left cerebral peduncle.

Growth protocol

All animal experimental protocols were approved by the Harvard University Institutional Animal Care and Use Committee, and complied with all relevant ethical regulations regarding animal research. Mice received food and water ad libitum, and were housed on a 12-hour on/off light cycle.

Extracted molecule

total RNA

Extraction protocol

nanoRibo-seq: RPFs were gel purified, then converted into libraries using the QiA-seq miRNA kit.
We performed neuronal FACS using established approaches (Catapano et al., 2001), but with the addition of translational inhibitors to prevent ex vivo elongation. For the CPN sorts, all buffers contained 100 ug/mL cycloheximide to inhibit translational elongation. We dissected brain into HBSS buffer+CHX, visualized the labeled region of cortex using an epifluorescent dissection microscope, and dissected the labeled cortical region into Disassociation Solution (DS)+CHX. We washed cortex tissue pieces twice in DS+CHX, then enzymatically digested twice by incubation in Enzyme Solution (DS with cysteine and papain)+CHX for 15 minutes each time (with inversion every 5 minutes to keep tissue pieces mixed). We washed twice in Wash Solution (WS)+CHX, triturated 15-20 times in ~1 mL of WS+CHX using fire-polished glass pipettes. We diluted the cell suspension with 4 mL WS+CHX, spun down cells for 5 minutes at 1000g, triturated again in 1 mL WS+CHX, and passed the cell suspension through a strainer cap. We added 1:1000 SYTOX Blue to be able to screen out dead cells. We then FACS-sorted the red cells, and collected them in a DNA LoBind Eppendorf Tube containing 2X polysome buffer+CHX (25 mM HEPES pH=8.0, 200 mM KCl, 10 mM MgCl2, 4 mM CaCl2, 1% nonidet P-40, 100 ug/mL CHX). For the SCPN sort, we performed all steps as described above, except that we omitted CHX until the SCPN were sorted into 2X polysome buffer+CHX.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

Ribo-seq

Data processing

To enable duplicate removal from these variable-length Ribo-seq sequences, we took advantage of the fact that we used paired-end sequencing, and that the first 12-nt of Read 2 of QiA-seq miRNA libraries is a 12-nt random barcode. We filtered for all reads with Read 2 > 12 reads (the vast majority), and extracted this random barcode using umi-tools: umi_tools extract --extract-method=string --bc-pattern=NNNNNNNNNNNN.
To remove the QiASeq miRNA adapters from the 3’ ends of Read 1, we ran cutadapt v2.8.: cutadapt -a AACTGTAGGCACCATCAAT
We then aligned Read 1 to the reference mouse rRNA sequences using STAR v2.6: STAR --runMode alignReads --runThreadN 8 --outSAMtype BAM Unsorted --readFilesCommand gunzip -c --outReadsUnmapped Fastx --outSAMmultNmax 1 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 21.
We then used the *final.out files produced by STAR for accounting the number and fraction of reads aligned to rRNA. We then aligned the reads that failed to align to rRNA to the mm10 genome, and to the mm10 transcriptome for analysis of footprint length distribution, coverage over coding sequence, and producing gene-level raw counts. We aligned to the mm10 genome (GRCm38 build) using STAR v2.6 and the following parameters:STAR --sjdbGTFfile Mus_musculus.GRCm38.95_chrNamed_headFix.gtf --runMode alignReads --runThreadN 8 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM --readFilesCommand gunzip -c --outSAMmultNmax 1 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 21
We completed duplicate removal following mm10 alignment to collapse all reads with the same alignment coordinates and exactly matching barcode sequences using umi-tools: umi_tools dedup --method=unique
We used the duplicate-removed bamFiles produced by this alignment for visualization, and for all further analysis. We then obtained raw, gene level counts over UTRs, CDS, and non-coding sequence using subRead:featureCounts using the annotations in Mus_musculus.GRCm38.95.gtf. We calculated coverage over 5’, 3’ UTRs and CDS as reads per kilobase per million mapped in Excel. We used custom shell scripts (makeLengthDistrosOverFeatures.sh, makeLengthDistrosOverNonTrans.sh) to compute the length distributions of the aligned reads from the lengths of aligned sequences.
Assembly: mm10, hg38
Supplementary files format and content: read_count_matrix: read_count matrix for reads mapping to gene CDS (CDS), 3'UTRs (three_utr), or 5' UTRs (five_utrs) obtained by subRead:featureCounts. read_length_distribution: read length distributions computed by custom shell script for reads mapping to CDS, three_utrs, five_utrs, snRNAs, snoRNAs, and lncRNAs. ORFs_collapsed.txt: text file with full information for all ORFs identified by RiboCode. ORFs_collapsed.bed: mm10 .bed file for all ORFs annotated by RiboCode. TPM.csv: Transcripts per Million Mapped for genes passing filter

Submission date

Jun 05, 2023

Last update date

Aug 25, 2023

Contact name

John Edward Froberg

E-mail(s)

jfroberg@fas.harvard.edu

Phone

8479770174

Organization name

Harvard University