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Sample GSM5259569

Query DataSets for GSM5259569

Status

Public on Apr 23, 2021

Title

Polysome selection pool 2 input cycle 0

Sample type

SRA

Source name

Cell culture

Organism

Homo sapiens

Characteristics

tissue: HEK293T cell line

Extracted molecule

total RNA

Extraction protocol

Transfected RNA was isolated and fractionated by sucrose gradient as in the "polysome fraction" samples. Fractions 10-16 were pooled and RNA was purified by acidic phenol chloroform extraction followed by silica column purification (Zymo)
1.5 µg RNA in 5.5 µL was mixed with 0.5 µL 2uM RT_Nluc26_UMI12_Read1Partial (CCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGGAACATCGTATGGGTAAACGGCCAT) and 0.5 µL 10 mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 3.5 µL reverse transcription mix was added to 10 µL total reaction volume: 2 µL 5x Superscript IV buffer, 0.5 µL 10 mM DTT, 0.5 µL Superase-In (ThermoFisher Scientific, AM2694), 0.5 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. For sequencing library preparation the RT reaction was PCR amplified under the following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10 µM Read1, 1 µL 10 µM Read2Partial_HBB29 in 20 µL total reaction volume. Cycling conditions were as follows: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec, 72°C for 10 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL first round PCR reaction, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10uM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1µL 10uM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions are: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 10 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads Beckman Coulter B23317).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Data processing

Library strategy: Barcode amplicon sequencing
Following adapter trimming, 670440 sequences with at least 10 summed read count across all libraries combined were set as the reference. Each library was aligned to this indexed reference using Bowtie2. Only uniquely mapping reads with edit distance ≤3 were retained. Alignments were further deduplicated using UMIcollapse (-p 0.05, -k 1). This results in the matrix of read count where rows are different sequence variants and columns are the samples. Normalized counts were obtained by dividing the matrix column-wise by total read counts per sample. For sequence variants with at least 15 reads in any one of the samples, a regression model was fitted on normalized read counts with the sequential selection rounds as ordinal predictors, penalizing differences between coefficients of adjacent groups (R package ordPens) . False discovery rate was estimated by Benjamini-Hochberg procedure. For choosing the final set of candidates, the criteria of ≥15 read counts in the final round polysome selection library and ≥2 fold enrichment over input in the final round was also required. For analysis of k-mers, 1 million reads (prior to alignment and analysis for highly enriched individual sequences) were sampled from each library. Position-specific k-mers were counted and statistical significance of pair-wise enrichments for each position-specific k-mers are calculated using kpLogo. The parameters were: zero-order Markov model background; 2≤k≤6; -shift 0 and -max-shift 0; binomial test with Bonferroni correction.
processed data files format and content: Table S3 contains k-mer enrichment statistics enriched after polysome selection for both input pools.

Submission date

Apr 22, 2021

Last update date

Apr 23, 2021

Contact name

Rhiju Das

E-mail(s)

[email protected]

Organization name

Stanford University School of Medicin

Department

Biochemistry