Diversity of ribosomes at the level of rRNA variation associated with human health and disease

. 2024 Aug 6;4(9):100629. doi: 10.1016/j.xgen.2024.100629

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Glass-bottom 12-well plates	Mattek	Cat# P12G-1.5-14-F
Bind-silane	GE Healthcare	Cat# 17-1330-01
Ethanol	VWR	Cat# 89125-170
Acetic acid	Sigma-Aldrich	Cat# A6283-100ML
Poly-D-lysine	Thermo Fisher Scientific	Cat# A3890401
Circular cover glass (12mm)	Electron Microscope Sciences	Cat# 72226-01
Gel slick solution	Lonza	Cat# 50640
1x PBS	Thermo Fisher Scientific	Cat# 10010049
Paraformaldehyde (PFA)	Electron Microscope Sciences	Cat# 15710-S
Methanol	Sigma-Aldrich	Cat# 34860-1L-R
Tween 20	Calbiochem	Cat# 655206
RNaseOUT	Thermo Fisher Scientific	Cat# 10777019
Ultrapure DNA/RNase-free water	Thermo Fisher Scientific	Cat# 10977023
Template switching RT enzyme mix	New England Biolabs	Cat# M0466L
dNTP mix	Invitrogen	Cat# 100004893
5-(3-aminoallyl)-dUTP	Invitrogen	Cat# AM8439
BS(PEG)₉	Thermo Fisher Scientific	Cat# 21582
Glycine	Sigma-Aldrich	Cat# 50046-250G
RNase H	New England Biolabs	Cat# M0297L
RNase A	Thermo Fisher Scientific	Cat# EN0531
RNase T1	Thermo Fisher Scientific	Cat# EN0541
BSA, molecular biology grade	New England Biolabs	Cat# B9000S
T4 DNA ligase	Thermo Fisher Scientific	Cat# EL0011
Phi29 DNA polymerase	Thermo Fisher Scientific	Cat# EP0094
Methacrylic acid N-hydroxysuccinimide ester	Sigma-Aldrich	Cat# 730300-1G
Sodium bicarbonate	Sigma-Aldrich	Cat# S5761-500G
Acrylamide solution	Bio-Rad	Cat# 161-0140
Bis-acrylamide solution	Bio-Rad	Cat# 161-0142
20x SSC	Sigma-Aldrich	Cat# S6639
Ammonium persulfate	Sigma-Aldrich	Cat# A3678
N,N,N,N -Tetramethylethylenediamine	Sigma-Aldrich	Cat# T9281
Formamide	Calbiochem	Cat# 75-12-7
Triton X-100	Sigma-Aldrich	Cat# 93443
4′,6-diamidino-2-phenylindole (DAPI)	Sigma-Aldrich	Cat# D9542
Fetal Bovine Serum	Gibco	Cat# 26140079
Trypsin-EDTA (0.5%), no phenol red	Gibco	Cat# 15400-054
DMEM/F12	Gibco	Cat# 11320033
Accutase	Gibco	Cat# A1110501
mTeSR1	StemCell Technologies	Cat# 85850
Thiazovivin	Tocris	Cat# 3845
Bicine	Fisher Scientific	Cat# ICN10100580
DMS	Sigma-Aldrich	Cat# D186309
100% Ethanol	Gold Shield Distributors	Cat# 0412804-PINT
2-Mercaptoethanol	Sigma-Aldrich	Cat# 2-Mercaptoethanol
TRIzol™ reagent	Invitrogen	Cat# 15596026
Chloroform	Fisher Scientific	Cat# C298-500
Zymo RNA Clean and Concentrator Kit-5	Zymo Research	Cat# R1016
TURBO™ DNase	Ambion	Cat# AM2238
SUPERase In RNase Inhibitor	Ambion	Cat# AM2696
Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (100X)	Thermo Fisher Scientific	Cat# 78443
Sucrose	Millipore	Cat# 8510-OP
10% SDS	Invitrogen	Cat# AM9820
Sodium acetate	Invitrogen	Cat# AM9740
Acid-phenol:chloroform, pH 4.5 with IAA (125:24:1)	Invitrogen	Cat# AM9722
TGIRT-III	InGex	Cat# TGIRT50
Buffer Kit, RNase-free	Invitrogen	Cat# AM9010
PEG 8000	Promega	Cat# V3011
DTT (Dithiothreitol)	Thermo Fisher Scientific	Cat# A39255
Betaine, 5M Solution	Fisher Scientific	Cat# AAJ77507UCR
Hydrochloric acid	Fisher Scientific	Cat# AA33257P6
Sodium hydroxide	Fisher Scientific	Cat# SS255-1
SPRIselect beads	Beckman Coulter	Cat# B23319
Protein LoBind tubes	Eppendorf	Cat# 0030108442
Buffer EB	Qiagen	Cat# 19086
Exo-Resistant Random Primer	Thermo Fisher Scientific	Cat# SO181
NEBNext® Ultra™ II Non-Directional RNA Second Strand Synthesis Module	New England Biolabs	Cat# E6111S
SMRTbell prep kit 3.0	PacBio	Cat# 102-182-700

Experimental models: cell lines

H7-hESC	Thomson et al.⁶³	N/A
K-562	ATCC	Cat# CCL-243
HeLa	ATCC	Cat# CCL-2

Oligonucleotides

28S RT primer bc11 for K-562 heavy polysome: CTATACGTATATCTATgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc12 for K-562 medium polysome: ACACTAGATCGCGTGTgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc13 for K-562 light polysome: CTCTCGCATACGCGAGgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc14 for K-562 monosome: CTCACTACGCGCGCGTgacaaacccttgtgtcgagg	This paper	N/A
18S RT primer bc11 for K-562 heavy polysome: CTATACGTATATCTATtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc12 for K-562 medium polysome: ACACTAGATCGCGTGTtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc13 for K-562 light polysome: CTCTCGCATACGCGAGtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc14 for K-562 monosome: CTCACTACGCGCGCGTtaatgatccttccgcaggttc	This paper	N/A
28S RT primer bc1 for H7-hESC heavy polysome: CACATATCAGAGTGCGgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc2 for H7-hESC medium polysome: ACACACAGACTGTGAGgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc3 for H7-hESC light polysome: ACACATCTCGTGAGAGgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc4 for H7-hESC monosome: CACGCACACACGCGCGgacaaacccttgtgtcgagg	This paper	N/A
18S RT primer bc1 for H7-hESC heavy polysome: CACATATCAGAGTGCGtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc2 for H7-hESC medium polysome: ACACACAGACTGTGAGtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc3 for H7-hESC light polysome: ACACATCTCGTGAGAGtaatgatccttccgcaggttc	This paper	N/A
18S RT primer bc4 for H7-hESC monosome: CACGCACACACGCGCGtaatgatccttccgcaggttc	This paper	N/A
28S RT primer bc6 H7-hESC: CATATATATCAG CTGTgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc20 H7-hESC: CACGACACGA CGATGTgacaaacccttgtgtcgagg	This paper	N/A
28S RT primer bc01 for DMS seq:/5Phos/CAC ATATCAGAGTGCGgacaaacccttgtgtcgagg	This paper	N/A

Deposited data

Monosome and polysome sequencing of 18S and 28S from monosome and polysome fractions from H7-hESC cell line	This paper	BioProject ID PRJNA926787, SRA: SRR29419059 (monosomes), SRA: SRR29419058 (“light polysomes” with 2 or 3 ribosomes), SRA: SRR29419057 (“medium polysomes” with 4 or 5 ribosomes), SRA: SRR29419056 (“heavy polysomes” with 6 or more ribosomes)
Monosome and polysome sequencing of 18S and 28S from monosome and polysome fractions from K562 cell line	This paper	BioProject ID PRJNA926787, SRA: SRR29419055 (monosomes), SRA: SRR29419054 (“light polysomes” with 2 or 3 ribosomes), SRA: SRR29419053 (“medium polysomes” with 4 or 5 ribosomes), SRA: SRR29419052 (“heavy polysomes” with 6 or more ribosomes)
Whole-genome sequencing of H7-hESC	This paper	BioProject ID PRJNA926787, SRA: SRR23196516
28S rRNA sequencing after treatment with DMS in H7-hESC cell line	This paper	BioProject ID PRJNA926787, SRA: SRR29884466

Software and algorithms

calc_word.py	Zielezinski et al.⁶⁴	Alfree tools (pip install alfpy) version 1.0.6
Bowtie2	Langmeade and Salzberg⁶⁵	Bowtie2 2.3.4.1
Clustal Omega	Sievers et al.⁶⁶	Clustal Omega - 1.2.4
Kallisto	Bray et al.⁶⁷	Kallisto 0.46.1
Minimap2	Li, H.⁶⁸	Minimap 2.17-r974-dirty
RGA method	This paper	Zenodo: https://zenodo.org/doi/10.5281/zenodo.11661415
Samtools	Li et al.⁶⁹	Samtools 1.16.1